Glucagon antagonists

ABSTRACT

Provided herein are compounds, including enantiomerically pure forms thereof, and pharmaceutically acceptable salts or co-crystals and prodrugs thereof which have glucagon receptor antagonist or inverse agonist activity. Further, provided herein are pharmaceutical compositions comprising the same as well as methods of treating, preventing, delaying the time to onset or reducing the risk for the development or progression of a disease or condition for which one or more glucagon receptor antagonist is indicated, including Type I and II diabetes, insulin resistance and hyperglycemia. Moreover, provided herein are methods of making or manufacturing compounds disclosed herein, including enantiomerically pure forms thereof, and pharmaceutically acceptable salts or Co-crystals and prodrugs thereof.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/058,604 which was filed Oct. 6, 2011, which is a371 of international application PCT/US2009/053795 filed Aug. 13, 2009,which claims priority to and the benefit of U.S. Provisional Application61/088,697 filed Aug. 13, 2008, the entire contents of all of which areincorporated herein by reference.

FIELD

Provided are antagonists of glucagon receptors. In particular, providedare Compounds and compositions for use in treatment, prevention oramelioration of one or more symptoms, causes, or effects of aglucoregulatory or glucagon receptor-mediated disease or disorder.

BACKGROUND

Glucagon is a 29-amino acid pancreatic hormone which is secreted fromthe pancreatic a cells into the portal blood supply in response tohypoglycemia. It acts as a counterregulatory hormone to insulin. Most ofthe physiological effects of glucagon are mediated by its interactionwith glucagon receptors in the liver, followed by activation ofadenylate cyclase to increase intracellular cAMP levels. The result isan increase in glycogenolysis and gluconeogenesis, while attenuating theability of insulin to inhibit these metabolic processes (Johnson et al.,J. Biol. Chem. 1972, 247, 3229-3235). As such, overall rates of hepaticglucose synthesis and glycogen metabolism are controlled by the systemicratio of insulin and glucagon (Roden et al., J. Clin. Invest. 1996, 97,642-648; Brand et al., Diabetologia 1994, 37, 985-993).

Diabetes is a disease characterized by elevated levels of plasmaglucose. Uncontrolled hyperglycemia is associated with an increased riskfor microvascular and macrovascular diseases, including nephropathy,retinopathy, hypertension, stroke, and heart disease. Control of glucosehomeostasis is a major approach to the treatment of diabetes. It hasbeen demonstrated in healthy animals as well as in animal models oftypes I and II diabetes that removal of circulating glucagon withselective and specific antibodies results in reduction of the glycemiclevel (Brand et al., Diabetologia 1994, 37, 985-993; Brand et al.,Diabetes 1994, 43 (Suppl. 1), 172A). Therefore, one of the potentialtreatments for diabetes and other diseases involving impaired glycemiais to block a glucagon receptor with a glucagon receptor antagonist toimprove insulin responsiveness, to decrease the rate of gluconeogenesis,and/or to lower plasma glucose levels by reducing the rate of hepaticglucose output in a patient.

Glucagon antagonists are known, e.g., UA20040014789, UA20040152750A1,WO04002480A1, U.S. Ser. No. 06/881,746B2, WO03053938A1, UA20030212119,UA20030236292, WO03048109A1, WO03048109A1, WO00069810A1, WO02040444A1,U.S. Ser. No. 06/875,760B2, UA20070015757A, WO04050039A2,UA20060116366A1, WO04069158A2, WO05121097A2, WO05121097A2, WO07015999A2,UA20070203186A1, UA20080108620A1, UA20060084681 A1, WO04100875A2,WO05065680A1 UA20070105930A1, U.S. Ser. No. 07/301,036B2,UA20080085926A1, WO08042223A1, WO07047177A1, UA20070088071A1,WO07111864A2, WO06102067A1, WO07136577A2, WO06104826A2, WO05118542A1,WO05123668A1, WO06086488, WO07106181A2, WO07114855A2, UA20070249688A1,WO07123581A1, WO06086488A2, WO07120270A2, WO07120284A2, andUA20080125468A1, although at this time none are commercially availableas therapeutics. Not all compounds that are glucagon antagonists havecharacteristics affording the best potential to become usefultherapeutics. Some of these characteristics include high affinity at theglucagon receptor, duration of receptor activation, oralbioavailability, and stability (e.g., ability to formulate orcrystallize, shelf life). Such characteristics can lead to improvedsafety, tolerability, efficacy, therapeutic index, patient compliance,cost efficiency, manufacturing ease, etc. It has been unexpectedlydiscovered that specific stereochemistry and functional groups of thecompounds of the present invention exhibit one or more of these desiredcharacteristics, including markedly improved receptor bindingproperties, oral bioavailability, and/or other advantageous featuresthat enhance their suitability for therapeutic use.

All documents referred to herein are incorporated by reference into thepresent application as though fully set forth herein.

BRIEF SUMMARY

Provided herein are compounds, including enantiomerically pure formsthereof, and pharmaceutically acceptable salts or co-crystals andprodrugs thereof which have glucagon receptor antagonist or inverseagonist activity. Further, provided herein are pharmaceuticalcompositions comprising the same, as well as methods of treating,preventing, delaying the time to onset or reducing the risk for thedevelopment or progression of a disease or condition for which one ormore glucagon receptor antagonist is indicated, including withoutlimitation Type I and II diabetes, insulin resistance and hyperglycemia.Moreover, provided herein are methods of making or manufacturingcompounds disclosed herein, including enantiomerically pure formsthereof, and pharmaceutically acceptable salts or co-crystals andprodrugs thereof.

By resolution of a synthetic intermediate into pure enantiomeric forms,a more active enantiomeric series was identified and was characterizedas having the R configuration. Methods for synthesis of theseenantiomers are described herein. No crystal structure was obtained,however, so while the depictions herein represent the R enantiomer, thecompounds of the invention are those that represent the most activeenantiomer and were obtained by the synthetic methods described herein.The most active enantiomers (compounds of the invention) are those thatdisplay improved characteristics, such as surprisingly increased ratioactivity vs. the S enantiomer at the cellular level as compared toactivity in the receptor displacement assay.

In one aspect, a compound of Formula I is provided:

wherein:

R⁴⁴ is H, CH₃ or CH₃CH₂; R⁴³ is C₁₋₆-alkyl, alkenyl, alkoxy,C₃₋₆-cycloalkyl, C₄₋₈-cycloalkenyl, C₄₋₈-bicycloalkenyl, aryl orheteroaryl, any of which can be optionally substituted with one or moresubstituents selected from C₁₋₆alkyl, CF₃, F, CN or OCF₃;

L is phenyl, indenyl, benzoxazol-2-yl, C₃₋₆-cycloalkyl,C₄₋₈-cycloalkenyl or C₄₋₈-bicycloalkenyl, any of which can be optionallysubstituted with one or more substituents selected from F, Cl, CH₃, CF₃,OCF₃ or CN; and

R⁴⁶ represents one or more substituents selected from H, F, Cl, CH₃,CF₃, OCF₃ or CN;

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In another aspect, a compound of Formula II is provided:

wherein:

R⁴⁴ is H;

R⁴⁵ is cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl,4,4-dimethylcyclohexyl, 4,4-diethylcyclohexyl, 4,4-dipropylcyclohexyl,4,4-diethylcyclohex-1-enyl 4,4-dimethylcyclohex-1-enyl,(S)-4-t-butylcyclohex-1-enyl, (R)-4-t-butylcyclohex-1-enyl,dipropylcyclohex-1-enyl, 4-t-butylphenyl,(1R,4S)-1,7,7-trimethylbicyclo[2.2.1]-3-hept-2-enyl or(1R,4R)-1,7,7-trimethylbicyclo[2.2.1]-2-hept-2-enyl;

L is phenyl, benzoxazol-2-yl, 4-alkyl-cyclohex-1-enyl,4,4-dialkylcyclohex-1-enyl, 4-alkyl-cyclohexyl, 4,4-dialkylcyclohexyl or4,4-dimethylcyclohexenyl, any of which can be optionally substitutedwith one or more substituents selected from F, Cl, CH₃, CF₃, OCF₃ or CN;and

R⁴⁶ is H;

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In another aspect, pharmaceutical compositions are provided comprising acompound provided herein, e.g., a compound of Formula I or Formula II,including a single enantiomer, a mixture of enantiomers, or a mixture ofdiastereomers thereof; or a pharmaceutically acceptable salt, solvate,or prodrug thereof in combination with one or more pharmaceuticallyacceptable carriers.

Further provided herein is a method of treating, preventing, orameliorating one or more symptoms of a condition, disorder, or diseaseassociated with a glucagon receptor, comprising administering to asubject having or being suspected to have such a condition, disorder, ordisease, a therapeutically effective amount of a compound providedherein, e.g., a compound of Formula I or II, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

Additionally, provided herein is a method of treating, preventing, orameliorating one or more symptoms of a condition, disorder, or diseaseresponsive to the modulation of a glucagon receptor, comprisingadministering to a subject having or being suspected to have such acondition, disorder, or disorder, a therapeutically effective amount ofa compound provided herein, e.g., a compound of Formula I or II, or apharmaceutically acceptable salt, solvate, or prodrug thereof; or apharmaceutical composition thereof.

Provided herein is a method of treating, preventing, or ameliorating oneor more symptoms of a GCGR-mediated condition, disorder, or disease,comprising administering to a subject having or being suspected to havesuch a condition, disorder, or disease, a therapeutically effectiveamount of a compound provided herein, e.g., a compound of Formula I orII, or a pharmaceutically acceptable salt, solvate, or prodrug thereofor a pharmaceutical composition thereof.

Provided herein is a method of treating, preventing, or ameliorating oneor more symptoms of a condition, disorder, or disease responsive to adecrease in the hepatic glucose production or in the blood glucose levelof a subject, comprising administering to the subject a therapeuticallyeffective amount of a compound provided herein, e.g., a compound ofFormula I or II, or a pharmaceutically acceptable salt, solvate, orprodrug thereof; or a pharmaceutical composition thereof.

Provided herein is a method of modulating the biological activity of aglucagon receptor, comprising contacting the receptor with one or moreof the compounds provided herein, e.g., a compound of Formula I or II,or a pharmaceutically acceptable salt, solvate, or prodrug thereof; or apharmaceutical composition thereof.

These and other aspects of the invention will be more clearly understoodwith reference to the following preferred embodiments and detaileddescription.

DETAILED DESCRIPTION a. Definitions

To facilitate understanding of the disclosure set forth herein, a numberof terms are defined below.

Generally, the nomenclature used herein and the laboratory procedures inorganic chemistry, medicinal chemistry, and pharmacology describedherein are those well known and commonly employed in the art. Unlessdefined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure belongs.

The term “subject” refers to an animal, including, but not limited to, aprimate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, rat,or mouse. The terms “subject” and “patient” are used interchangeablyherein in reference, for example, to a mammalian subject, such as ahuman subject.

The terms “treat,” “treating,” and “treatment” are meant to includealleviating or abrogating a disorder, disease, or condition, or one ormore of the symptoms associated with the disorder, disease, orcondition; or alleviating or eradicating the cause(s) of the disorder,disease, or condition itself.

The terms “prevent,” “preventing,” and “prevention” are meant to includea method of delaying and/or precluding the onset of a disorder, disease,or condition, and/or its attendant symptom(s); barring a subject fromacquiring a disease; or reducing a subject's risk of acquiring adisorder, disease, or condition.

The term “therapeutically effective amount” are meant to include theamount of a compound that, when administered, is sufficient to preventdevelopment of, or alleviate to some extent, one or more of the symptomsof the disorder, disease, or condition being treated. The term“therapeutically effective amount” also refers to the amount of acompound that is sufficient to elicit the biological or medical responseof a cell, tissue, system, animal, or human, which is being sought by aresearcher, veterinarian, medical doctor, or clinician.

The term “IC₅₀” refers an amount, concentration, or dosage of a compoundthat is required for 50% inhibition of a maximal response in an assaythat measures such response.

The term “pharmaceutically acceptable carrier,” “pharmaceuticallyacceptable excipient,” “physiologically acceptable carrier,” or“physiologically acceptable excipient” refers to apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent, or encapsulatingmaterial. In one embodiment, each component is “pharmaceuticallyacceptable” in the sense of being compatible with the other ingredientsof a pharmaceutical formulation, and suitable for use in contact withthe tissue or organ of humans and animals without excessive toxicity,irritation, allergic response, immunogenicity, or other problems orcomplications, commensurate with a reasonable benefit/risk ratio. See,Remington: The Science and Practice of Pharmacy, 21st Edition,Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook ofPharmaceutical Excipients, 5th Edition, Rowe et al., Eds., ThePharmaceutical Press and the American Pharmaceutical Association: 2005;and Handbook of Pharmaceutical Additives, 3rd Edition. Ash and Ash Eds.,Gower Publishing Company: 2007; Pharmaceutical Preformulation andFormulation, Gibson Ed., CRC Press Boca Raton, Fla., 2004.

The term “about” or “approximately” means an acceptable error for aparticular value as determined by one of ordinary skill in the art,which depends in part on how the value is measured or determined. Incertain embodiments, the term “about” or “approximately” means within 1,2, 3, or 4 standard deviations. In certain embodiments, the term “about”or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

The terms “active ingredient” and “active substance” refer to acompound, which is administered, alone or in combination with one ormore pharmaceutically acceptable excipients, to a subject for treating,preventing, or ameliorating one or more symptoms of a condition,disorder, or disease. As used herein, “active ingredient” and “activesubstance” may be an optically active isomer of a compound describedherein.

The terms “drug,” “therapeutic agent,” and “chemotherapeutic agent”refer to a compound, or a pharmaceutical composition thereof, which isadministered to a subject for treating, preventing, or ameliorating oneor more symptoms of a condition, disorder, or disease.

The term “naturally occurring” or “native” when used in connection withbiological materials such as nucleic acid molecules, polypeptides, hostcells, and the like, refers to materials which are found in nature andare not manipulated by man. Similarly, “non-naturally occurring” or“non-native” refers to a material that is not found in nature or thathas been structurally modified or synthesized by man.

The term “glucagon receptor” or “GCGR” refers to a glucagon receptorprotein or variant thereof, which is capable of mediating a cellularresponse to glucagon in vitro or in vivo, GCGR variants include proteinssubstantially homologous to a native GCGR, i.e., proteins having one ormore naturally or non-naturally occurring amino acid deletions,insertions, or substitutions (e.g., GCGR derivatives, homologs, andfragments), as compared to the amino acid sequence of a native GCGR. Incertain embodiments, the amino acid sequence of a GCGR variant is atleast about 80% identical, at least about 90% identical, or at leastabout 95% identical to a native GCGR. In certain embodiments, the GCGRis a human glucagon receptor.

The term “glucagon receptor antagonist” or “GCGR antagonist” refers to acompound that, e.g., partially or completely blocks, decreases,prevents, inhibits, or downregulates GCGR activity. These terms alsorefer to a compound that binds to, delays the activation of,inactivates, or desensitizes GCGR. A GCGR antagonist may act byinterfering with the interaction of glucagon with GCGR.

The term “GCGR-mediated condition, disorder, or disease” refers to acondition, disorder, or disease characterized by inappropriate, e.g.,less than or greater than normal, GCGR activity. Inappropriate GCGRfunctional activity might arise as the result of an increase in glucagonconcentration, GCGR expression in cells which normally do not expressGCGR, increased GCGR expression or degree of intracellular activation,leading to, e.g., abnormal plasma glucose levels; or decreased GCGRexpression. A GCGR-mediated condition, disorder or disease may becompletely or partially mediated by inappropriate GCGR activity. Inparticularly, a GCGR-mediated condition, disorder or disease is one inwhich modulation of GCGR results in some effect on the underlyingsymptom, condition, disorder, or disease, e.g., a GCGR antagonistresults in some improvement in at least some of patients being treated.

The term “alkyl” and the prefix “alk” refers to a linear or branchedsaturated monovalent hydrocarbon radical, wherein the alkyl mayoptionally be substituted with one or more substituents. The term“alkyl” also encompasses linear, branched, and cyclic alkyl, unlessotherwise specified. In certain embodiments, the alkyl is a linearsaturated monovalent hydrocarbon radical that has 1 to 20 (C1-20), 1 to15 (C1-15), 1 to 12 (C1-12), 1 to 10 (C1-10), or 1 to 6 (C1-6) carbonatoms, or branched saturated monovalent hydrocarbon radical of 3 to 20(C3-20), 3 to 15 (C3-15), 3 to 12 (C3-12), 3 to 10 (C3-10), or 3 to 6(C3-6) carbon atoms. As used herein, linear C1-6 and branched C3-6 alkylgroups are also referred as “lower alkyl.” Examples of alkyl groupsinclude, but are not limited to, methyl, ethyl, propyl (including allisomeric forms), n-propyl, isopropyl, butyl (including all isomericforms), n-butyl, isobutyl, t-butyl, pentyl (including all isomericforms), and hexyl (including all isomeric forms). For example, C1-6alkyl refers to a linear saturated monovalent hydrocarbon radical of 1to 6 carbon atoms or a branched saturated monovalent hydrocarbon radicalof 3 to 6 carbon atoms. Cycloalkyl also includes monocyclic rings fusedto an aryl group in which the point of attachment is on the non-aromaticportion. Examples of cycloalkyl include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl,decahydronaphthyl, indanyl and the like.

The term “alkenyl” refers to a linear or branched monovalent hydrocarbonradical, which contains one or more, in one embodiment, one to five,carbon-carbon double bonds. The alkenyl may be optionally substitutedwith one or more substituents. The term “alkenyl” also embraces radicalshaving “cis” and “trans” configurations, or alternatively, “E” and “Z”configurations, as appreciated by those of ordinary skill in the art. Asused herein, the term “alkenyl” encompasses both linear and branchedalkenyl, unless otherwise specified. For example, C2-6 alkenyl refers toa linear unsaturated monovalent hydrocarbon radical of 2 to 6 carbonatoms or a branched unsaturated monovalent hydrocarbon radical of 3 to 6carbon atoms. In certain embodiments, the alkenyl is a linear monovalenthydrocarbon radical of 2 to 20 (C2-20), 2 to 15 (C2-15), 2 to 12(C2-12), 2 to 10 (C2-10), or 2 to 6 (C2-6) carbon atoms, or a branchedmonovalent hydrocarbon radical of 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to12 (C3-12), 3 to 10 (C3-10), or 3 to 6 (C3-6) carbon atoms. Examples ofalkenyl groups include, but are not limited to, vinyl, isopropenyl,pentenyl, hexenyl, heptenyl, ethenyl, propen-1-yl, propen-2-yl, allyl,butenyl, 2-butenyl, 2-methyl-2-butenyl, 4-methylbutenyl, and the like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical, which contains one or more, in one embodiment, one to five,carbon-carbon triple bonds. The alkynyl may be optionally substitutedwith one or more substituents. The term “alkynyl” also encompasses bothlinear and branched alkynyl, unless otherwise specified. In certainembodiments, the alkynyl is a linear monovalent hydrocarbon radical of 2to 20 (C2-20), 2 to 15 (C2-15), 2 to 12 (C2-12), 2 to 10 (C2-10), or 2to 6 (C2-6) carbon atoms, or a branched monovalent hydrocarbon radicalof 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 12 (C3-12), 3 to 10 (C3-10),or 3 to 6 (C3-6) carbon atoms. Examples of alkynyl groups include, butare not limited to, ethynyl (—C≡CH), propargyl (—CH2C≡CH),3-methyl-1-pentynyl, 2-heptynyl, and the like. For example, C2-6 alkynylrefers to a linear unsaturated monovalent hydrocarbon radical of 2 to 6carbon atoms or a branched unsaturated monovalent hydrocarbon radical of3 to 6 carbon atoms.

The term “cycloalkyl” refers to a cyclic saturated bridged and/ornon-bridged monovalent hydrocarbon radical, which may be optionallysubstituted with one or more substituents. In certain embodiments, thecycloalkyl has from 3 to 20 (C3-20), from 3 to 15 (C3-15), from 3 to 12(C3-12), from 3 to 10 (C3-10), or from 3 to 7 (C3-7) carbon atoms.Examples of cycloalkyl groups include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,decalinyl, and adamantyl.

The term “cycloalkenyl” refers to a cyclic unsaturated bridged and/ornon-bridged monovalent hydrocarbon radical, which contains one or moredouble bonds in its ring. The cycloalkenyl may be optionally substitutedwith one or more substituents. In certain embodiments, the cycloalkenylhas from 3 to 20 (C3-20), from 3 to 15 (C3-15), from 3 to 12 (C3-12),from 3 to 10 (C3-10), or from 3 to 7 (C3-7) carbon atoms.

The term “aryl” (Ar) refers to a monocyclic aromatic group and/ormulticyclic monovalent aromatic group that contain at least one aromatichydrocarbon ring. In certain embodiments, the aryl has from 6 to 20(C6-20), from 6 to 15 (C6-15), or from 6 to 10 (C6-10) ring atoms.Examples of aryl groups include, but are not limited to, phenyl,naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl,and terphenyl. Aryl also refers to bicyclic or tricyclic carbon rings,where one of the rings is aromatic and the others of which may besaturated, partially unsaturated, or aromatic, for example,dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl).In certain embodiments, aryl may also be optionally substituted with oneor more substituents.

The term “aralkyl” or “aryl-alkyl” refers to a monovalent alkyl groupsubstituted with aryl. In certain embodiments, both alkyl and aryl maybe optionally substituted with one or more substituents.

The term “heteroaryl” (HAR) refers to a monocyclic aromatic group and/ormulticyclic aromatic group that contain at least one aromatic ring,wherein at least one aromatic ring contains one or more heteroatomsindependently selected from O, S, and N. In some embodiments, each ringcontains 5 to 6 atoms. Each ring of a heteroaryl group can contain oneor two O atoms, one or two S atoms, and/or one to four N atoms, providedthat the total number of heteroatoms in each ring is four or less andeach ring contains at least one carbon atom. The heteroaryl may beattached to the main structure at any heteroatom or carbon atom whichresults in the creation of a stable compound. In certain embodiments,the heteroaryl has from 5 to 20, from 5 to 15, or from 5 to 10 ringatoms. Examples of monocyclic heteroaryl groups include, but are notlimited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl,isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl,oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyramidyl, pyridazinyl,triazolyl, tetrazolyl, and triazinyl. Examples of bicyclic heteroarylgroups include, but are not limited to, indolyl, benzothiazolyl,benzoxazolyl, benzothienyl, benzothiophenyl, furo(2,3-b) pyridyl,quinolinyl, tetrahydroisoquinolinyl, isoquinolyl, isoquinolinyl,benzimidazolyl, benzopyranyl, indolizinyl benzofuranyl, isobenzofuranyl,chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl purinyl,pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, andtetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include,but are not limited to, carbazolyl, benzindolyl, phenanthrollinyl,acridinyl, phenanthridinyl, and xanthenyl. In certain embodiments,heteroaryl may also be optionally substituted with one or moresubstituents. Heteroaryl also includes aromatic heterocyclic groupsfused to heterocycles that are non-aromatic or partially aromatic, andaromatic heterocyclic groups fused to cycloalkyl rings. Heteroaryl alsoincludes such groups in charged form, e.g., pyridinium.

The term “heterocyclyl” (Hetcy) or “heterocyclic” refers to a monocyclicnon-aromatic ring system and/or multicyclic ring system that contains atleast one non-aromatic ring, wherein one or more of the non-aromaticring atoms are heteroatoms independently selected from O, S, or N; andthe remaining ring atoms are carbon atoms. In certain embodiments, theheterocyclyl or heterocyclic group has from 3 to 20, from 3 to 15, from3 to 10, from 3 to 8, from 4 to 7, or from 5 to 6 ring atoms. In certainembodiments, the heterocyclyl is a monocyclic, bicyclic, tricyclic, ortetracyclic ring system, which may include a fused or bridged ringsystem, and in which the nitrogen or sulfur atoms may be optionallyoxidized, the nitrogen atoms may be optionally quaternized, and somerings may be partially or fully saturated, or aromatic. The heterocyclylmay be attached to the main structure at any heteroatom or carbon atomwhich results in the creation of a stable compound. Examples of suchheterocyclic radicals include, but are not limited to benzoxazinyl,benzodioxanyl, benzodioxolyl, benzopyranonyl, benzopyranyl,benzotetrahydrofuranyl, benzotetrahydrothienyl, benzothiopyranyl,chromanyl, chromonyl, coumarinyl, decahydroisoquinolinyl,dihydrobenzisothiazinyl, dihydrobenzisoxazinyl,2,3-dihydrofuro(2,3-b)pyridyl, dihydrofuryl, dihydroindolyl,dihydropyranyl, dioxolanyl, dihydropyrazinyl, dihydropyridinyl,dihydropyrazolyl, dihydropyrimidinyl, dihydropyrrolyl, 1,4-dithianyl,imidazolidinyl, imidazolinyl, indolinyl, isobenzotetrahydrofuranyl,isobenzotetrahydrothienyl, isochromanyl, isocoumarinyl, isoindolinyl,isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl,octahydroisoindolyl, oxazolidinonyl, oxazolidinyl, oxiranyl,quinuclidinyl, tetrahydrofuryl, tetrahydrofuranyl,tetrahydrohydroquinolinyl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydrothienyl, thiamorpholinyl, thiazolidinyl, and 1,3,5-trithianyl.Heterocyclyl/heterocyclic also includes partially unsaturated monocyclicrings that are not aromatic, such as 2- or 4-pyridones attached throughthe nitrogen or N-substituted-(1H, 3H)-pyrimidine-2,4-diones(N-substituted uracils). Heterocyclyl/heterocyclic also includes suchmoieties in charged form, e.g., piperidinium. In certain embodiments,heterocyclyl/heterocyclic may also be optionally substituted with one ormore substituents.

The term “alkoxy” refers to an —OR radical, wherein R is, for example,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl,each as defined herein. When R is aryl, it is also known as aryloxy.Examples of alkoxy groups include, but are not limited to, methoxy,ethoxy, propoxy, n-propoxy, 2-propoxy, n-butoxy, isobutoxy, tert-butoxy,cyclohexyloxy, phenoxy, benzoxy, and 2-naphthyloxy. In certainembodiments, alkoxy may also be optionally substituted with one or moresubstituents.

The term “acyl” refers to a —C(O)R radical, wherein R is, for example,hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, orheterocyclyl, each as defined herein. Examples of acyl groups include,bin are not limited to, formyl, acetyl, propionyl, butanoyl,isobutanoyl, pentanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl,decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl,eicosanoyl, docosanoyl, myristoleoyl, palmitoleoyl, oleoyl, linoleoyl,arachidonoyl, benzoyl, pyridinyicarbonyl, and furoyl. In certainembodiments, acyl may also be optionally substituted with one or moresubstituents.

The term “halogen”, “halide” or “halo” (Halo) refers to fluorine,chlorine, bromine, and/or iodine.

The term “optionally substituted” is intended to mean that a group,including alkyl, alkoxy, acyl, alkyl-cycloalkyl, hydroxyalkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, aryl,aryloxy, aralkyl, aryl-alkenyl, aryl-alkynyl, heteroaryl,heteroarylalkyl heteroaryl-alkenyl, heteroaryl-alkynyl, andheterocyclyl, or acyl, may be substituted with one or more substituents,in one embodiment, one, two, three, four substituents, where in someembodiments each substituent is independently selected from the groupconsisting of cyano, halo, oxo, nitro, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₇ cycloalkyl, C₆₋₁₄ aryl, heteroaryl, heterocyclyl,—C(O)R^(e), —C(O)OR^(e), —C(O)NR^(f)R^(g), —C(NR^(e))NR^(f)R^(g),—OR^(e), —OC(O)R^(e), —OC(O)OR^(e), —(O)NR^(f)R^(g),—OC(═NR^(e))NR^(f)R^(g), —OS(O)R^(e), —OS(O)₂R^(e), —OS(O)NR^(f)R^(g),—OS(O)₂NR^(f)R^(g), —NR^(f)R^(g), —NR^(e)C(O)R^(f), NR^(e)C(O)OR^(f),—NR^(e)C(O)NR^(f)R^(g), NR^(e)C(═NR^(h))NR^(f)R^(g), —NR^(e)S(O)R^(f),—NR^(e)S(O)₂R^(f), —NR^(e)S(O)NR^(f)R^(g), —NR^(e)S(O)₂NR^(f)R^(g),—SR^(e), —S(O)R^(e), S—(O)₂R^(e), and —S(O)₂NR^(f)R^(g), wherein eachR^(e), R^(f), R^(g), and R^(h) is independently hydrogen, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ cycloalkyl, C₆₋₁₄ aryl, heteroaryl, orheterocyclyl; or R^(f) and R^(g) together with the N atom to which theyare attached form heterocyclyl.

The term “optically active” refers to a collection of molecules, whichhas an enantiomeric excess of no less than about 50%, no less than about70%, no less than about 80%, no less than about 90%, no less than about91%, no less than about 92%, no less than about 93%, no less than about94%, no less than about 95%, no less than about 96%, no less than about97%, no less than about 98%, no less than about 99%, no less than about99.5%, or no less than about 99.8%.

In describing an optically active compound, the prefixes R and S areused to denote the absolute configuration of the molecule about itschiral center(s). The (+) and (−) are used to denote the opticalrotation of the compound, that is, the direction in which a plane ofpolarized light is rotated by the optically active compound. The (−)prefix indicates that the compound is levorotatory, that is, thecompound rotates the plane of polarized light to the left orcounterclockwise. The (+) prefix indicates that the compound isdextrorotatory, that is, the compound rotates the plane of polarizedlight to the right or clockwise. However, the sign of optical rotation,(+) and (−), is not related to the absolute configuration of themolecule, R and S.

The term “solvate” refers to a compound provided herein or a saltthereof, which further includes a stoichiometric or non-stoichiometricamount of solvent bound by non-covalent intermolecular forces. Where thesolvent is water, the solvate is a hydrate.

“Binding” means the specific association of the compound of interest tothe target of interest, e.g., a receptor.

The term “crystalline” and related terms used herein, when used todescribe a substance, component or product, means that the substance,component or product is crystalline as determined by X-ray diffraction.See, e.g., Remington's Pharmaceutical Sciences, 18^(th) ed., MackPublishing, Easton Pa., 173 (1990); The United States Pharmacopeia,23^(rd) ed., 1843-1844 (1995).

“Co-crystal” as used herein means a crystalline material comprised oftwo or more unique solids at room temperature that are H-bonded.

“Diabetes” refers to a heterogeneous group of disorders that shareimpaired glucose tolerance in common. Its diagnosis andcharacterization, including pre-diabetes, type I and type II diabetes,and a variety of syndromes characterized by impaired glucose tolerance,impaired fasting glucose, and abnormal glycosylated hemoglobin, are wellknown in the art. It may be characterized by hyperglycemia, glycosuria,ketoacidosis, neuropathy or nephropathy, increased hepatic glucoseproduction, insulin resistance in various tissues, insufficient insulinsecretion and enhanced or poorly controlled glucagon secretion from thepancreas.

The term “drug” refers to a compound, or a pharmaceutical compositionthereof, which is administered to a subject for treating, preventing, orameliorating one or more symptoms of a condition, disorder, or disease.

The term “EC₅₀” refers an amount, concentration, or dosage of a compoundat which 50% of a maximal response is observed in an assay that measuressuch response.

The term “percent enantiomeric excess (% cc)” refers to optical purity.It is obtained by using the following formula:

${\frac{\lbrack R\rbrack - \lbrack S\rbrack}{\lbrack R\rbrack + \lbrack S\rbrack} \times 100} = {{\%\mspace{14mu} R} - {\%\mspace{14mu} S}}$

where [R] is the amount of the R isomer and [S] is the amount of the Sisomer. This formula provides the % ee when R is the dominant isomer.

The term “enantiomerically pure” refers to a compound which comprises atleast about 80% by weight of the designated enantiomer and at most about20% by weight of the other enantiomer or other stereoisomer(s), at leastabout 90% by weight of the designated enantiomer and at most about 10%by weight of the other enantiomer or other stereoisomer(s), at leastabout 95% by weight of the designated enantiomer and at most about 5% byweight of the other enantiomer or other stereoisomer(s), at least about96.6% by weight of the designated enantiomer and at most about 3.4% byweight of the other enantiomer or other stereoisomer(s), at least about97% by weight of the designated enantiomer and at most about 3% byweight of the other enantiomer or other stereoisomer(s), at least about99% by weight of the designated enantiomer and at most about 1% byweight of the other enantiomer or other stereoisomer(s), or at leastabout 99.9% by weight of the designated enantiomer and at most about0.1% by weight of the other enantiomer or other stereoisomer(s). Incertain embodiments, the weights are based upon total weight of thecompound.

As used in connection with the compounds of Formula I and II disclosedherein, the terms “R-isomer” and “R-enantiomer” refer to theconfiguration R of the aliphatic carbon which is alpha to the +C(O)NH—group. Formula I below shows the R-stereochemistry.

The term “chiral” as used herein includes a compound that has theproperty that it is not superimposable on its mirror image.

“Insulin resistance” is defined clinically as the impaired ability of aknown quantity of exogenous or endogenous insulin to increase whole bodyglucose uptake and utilization.

“Impaired glucose tolerance (IGT)” refers to a condition known toprecede the development of overt Type 2 diabetes. It is characterized byabnormal blood glucose excursions following a meal. The criteria fordiagnosing and characterizing impaired glucose tolerance and relatedsyndromes are well known in the art.

“Lower” referred to herein in connection with organic radicals orcompounds respectively defines such radicals or compounds as containingup to and including 6 carbon atoms. One aspect provides organic radicalsor compounds as containing up to and including 4 carbon atoms. Yetanother aspect provides organic radicals or compounds that contain oneto three carbon atoms. Such groups may be straight chain, branched, orcyclic.

“Metabolic disease” includes diseases and conditions such as obesity,diabetes and lipid disorders such as hypercholesterolemia,hyperlipidemia, hypertriglyceridemia as well as disorders that areassociated with abnormal levels of lipoproteins, lipids, carbohydratesand insulin such as metabolic syndrome X, diabetes, impaired glucosetolerance, atherosclerosis, coronary artery disease, cardiovasculardisease. The criteria for diagnosing and characterizing these conditionsand related syndromes are well known in the art.

“Prodrug” as used herein refers to any compound that when administeredto a biological system generates a biologically active compound as aresult of spontaneous chemical reaction(s), enzyme catalyzed chemicalreaction(s), and/or metabolic chemical reaction(s), or a combination ofeach. Standard prodrugs are formed using groups attached tofunctionality, e.g., HO—, HS—, HOOC—, —NHR, associated with the drug,that cleave in vivo. Standard prodrugs include but are not limited tocarboxylate esters where the group is alkyl, aryl, aralkyl,acyloxyalkyl, alkoxycarbonyloxyalkyl as well as esters of hydroxyl;thiol and amines where the group attached is an acyl group, analkoxycarbonyl, aminocarbonyl, phosphate or sulfate. The groupsillustrated are exemplary, not exhaustive, and one skilled in the artcould prepare other known varieties of prodrugs. Such prodrugs of thecompounds of Formula I or II disclosed herein fall within this scope.Prodrugs must undergo some form of a chemical transformation to producethe compound that is biologically active or is a precursor of thebiologically active compound. In some cases, the prodrug is biologicallyactive, usually less than the drug itself, and serves to improve drugefficacy or safety through improved oral bioavailability, and/orpharmacodynamic half-life, etc. Prodrug forms of compounds may beutilized, for example, to improve bioavailability, improve subjectacceptability such as by masking or reducing unpleasant characteristicssuch as bitter taste or gastrointestinal irritability, alter solubilitysuch as for intravenous use, provide for prolonged or sustained releaseor delivery, improve ease of formulation, or provide site-specificdelivery of the compound. Prodrugs are described in The OrganicChemistry of Drug Design and Drug Action, by Richard B. Silverman,Academic Press, San Diego, 1992, Chapter 8: “Prodrugs and Drug deliverySystems” pp. 352-401; Design of Prodrugs, edited by H. BundgaardElsevier Science, Amsterdam, 1985; Design Biopharmaceutical Propertiesthrough Prodrugs and Analogs, Ed. by F. B. Roche, AmericanPharmaceutical Association, Washington, 1977; and Drug Delivery Systems,ed. by R. L. Juliano, Oxford Univ. Press, Oxford, 1980.

b. Compounds

One aspect provides for compounds of Formula I

wherein;

R⁴⁴ is H, CH₃ or CH₃CH₂;

R⁴⁵ is C₁₋₆-alkyl, alkenyl, alkoxy, C₃₋₆-cycloalkyl, C₄₋₈-cycloalkenyl,C₄₋₈-bicycloalkenyl, aryl or heteroaryl, any of which can be optionallysubstituted with one or more substituents selected from C₁₋₆alkyl, CF₃,F, CN or OCF₃;

L is phenyl, indenyl, benzoxazol-2-yl or 4,4-dimethylcyclohexenyl, anyof which can be optionally substituted with one or more substituentsselected from F, Cl, CH₃, CF₃, OCF₃ or CN; and

R⁴⁶ represents one or more substituents selected from H, F, Cl, CH₃,CF₃, OCF₃ or CN.

In certain embodiments according to Formula I, the configuration of thealiphatic carbon which is alpha to the —C(O)NH— group is R.

In other embodiments according to Formula I, L is substituted with oneor more substituents independently selected from F, Cl, CH₃, CF₃, OCF₃or CN. In another embodiment, L is phenyl, benzoxazol-2-yl or4,4-dimethylcyclohexenyl, any of which can be optionally substitutedwith one or more substituents. In another embodiment, L is4-chloro-2-methylphenyl 4-methyl-2-benzoxazolyl, 2,4,6-trimethylphenyl,benzoxazol-2-yl, 4-chloro-3-methylphenyl or 4,4-dimethylcyclobexenyl.

In another embodiment, R⁴⁴ is 1-1 or CH₃. In another embodiment, R⁴⁴ isH.

In certain embodiments, R⁴⁵ is attached to the 3 (meta) or 4 (para)position. In another embodiment, R⁴⁵ is attached to the 4 (para)position. In another embodiment, R⁴ is alkenyl, C₃₋₆-cycloalkyl,C₄₋₈-cycloalkenyl, C₄₋₈-bicycloalkenyl or phenyl, any of which can beoptionally substituted with one or more substituents. In yet otherembodiments according to Formula I, R⁴⁵ is selected from (CH₃)₃CCH═CH—,t-butyl-cycloalkyl-, dimethyl-cycloalkyl-, t-butyl-cycloalkenyl-,dimethyl-cycloalkenyl-, bicycloalkenyl or phenyl-.

In certain embodiments according to Formula I, R⁴⁵ is substituted withone or more substituents independently selected from CH₃ and (CH₃)₃C—.

In certain embodiments according to Formula I, R⁴⁵ istrans-t-butylvinyl, cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl,4,4-dimethylcyclohexyl, cyclohex-1-enyl, (S)-4-t-butylcyclohex-1-enyl,(R)-4-t-butylcyclohex-1-enyl, 4,4-dimethylcyclohex-1-enyl,4,4-diethylcyclohex-1-enyl, 4,4-diethylcyclohexyl,4,4-dipropylcyclohex-1-enyl, 4,4-dipropylcyclohexyl,4,4-dimethylcyclohexa-1,5-dienyl,(1R,4S)-1,7,7-trimethylbicyclo[2.2.1]3-heptyl-2-ene,(1R,4R)-1,7,7-trimethylbicyclo[2.2.1]2-heptyl-2-ene,2-methyl-4-chloro-phenyl, 2,4,6-trimethylphenyl or 4-t-butylphenyl.

In certain embodiments according to Formula I, R⁴⁵ istrans-t-butylvinyl, cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl,4,4-dimethylcyclohexyl, (S)-4-t-butylcyclohex-1-enyl,(R)-4-t-butylcyclohex-1-enyl, 4,4-dimethylcyclohex-1-enyl,(1R,4R)-1,7,7-trimethylbicyclo[2.2.1]2-heptyl-2-ene or 4-t-butylphenyl.

In another embodiment, R⁴⁶ is H or CH₃. In another embodiment, R⁴⁶ is H.

In certain embodiments, the compound of Formula I is one presented belowin Table I:

TABLE 1 Certain Compounds of Formula I

Another aspect provides for compounds, prodrugs thereof, andcompositions comprising the compounds or prodrugs thereof wherein thecompound is a structure of Formula II:

wherein:

R⁴⁴ is H;

R⁴⁵ is cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl,4,4-dimethylcyclohexyl, 4,4-diethylcyclohexyl, 4,4-dipropylcyclohexyl,4,4-diethylcyclohex-1-enyl, 4,4-dimethylcyclohex-1-enyl,(S)-4-t-butylcyclohex-1-enyl, (R)-4-t-butylcyclohex-1-enyl,4,4-dipropylcyclohex-1-enyl, 4-t-butylphenyl,(1R,4S)-1,7,7-trimethylbicyclo[2.2.1]-3-hept-2-enyl or(1R,4R)-1,7,7-trimethylbicyclo[2.2.1]-2-hept-2-enyl;

L is phenyl, benzoxazol-2-yl, 4-alkyl-cyclohex-1-enyl,4,4-dialkylcyclohex-1-enyl, 4-alkyl-cyclohexyl, 4,4-dialkylcyclohexyl or4,4-dimethylcyclohexenyl, any of which can be optionally substitutedwith one or more substituents selected from F, Cl, CH₃, CF₃, OCF₃ or CN;and

R⁴⁶ is H.

In certain embodiments according to Formula II, L is substituted withone or more substituents independently selected from Cl or CH₃. Inanother embodiment, L is phenyl, benzoxazol-2-yl or4,4-dimethylcyclohexenyl, any of which can be optionally substitutedwith one or more substituents selected from Cl or CH₃.

In certain embodiments according to Formula II, R⁴⁵ is attached to the 3(meta) or 4 (para) position. In another embodiment, R⁴⁵ is attached tothe 4 (para) position.

In another embodiment, R⁴⁵ is cis-4-t-butylcyclohexyl,trans-4-t-butylcyclohexyl, 4,4-dimethylcyclohexyl,(S)-4-t-butylcyclohex-1-enyl, (R)-4-t-butylcyclohex-1-enyl,4,4-dipropylcyclohex-1-enyl, 4-t-butylphenyl,(1R,4S)-1,7,7-trimethylbicyclo[2.2.1]-3-hept-2-enyl or(1R,4R)-1,7,7-trimethylbicyclo[2.2.1]-2-kept-2-enyl. In otherembodiments, R⁴ is cis-4-t-butylcyclohexyl, 4,4-dimethylcyclohexyl,(S)-4-t-butylcyclohex-1-enyl, (R)-4-t-butylcyclohex-1-enyl,4-t-butylphenyl or (1R,4S)-1,7,7-trimethylbicyclo[2.2.1]-3-hept-2-enyl.

In certain embodiments the compound of Formula II is also a compound ofFormula I.

In certain embodiments according to Formula II, the configuration of thealiphatic carbon which is alpha to the —C(O)NH— group is R.

In certain embodiments, the compounds of Formula II are a racemicmixture.

Another aspect provides for enantiomerically pure compounds of Formula Ior II. In certain embodiments, a single enantiomeris >60%, >70%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%or >99% as compared to the total percentage of all other enantiomers ofthe same compound or other diastereomers present in the composition.

Another aspect provides enantiomerically pure compounds of Formula I orII. In certain embodiments, the compound comprises at least about 80% byweight of the designated enantiomer and at most about 20% by weight ofthe other enantiomer or other stereoisomer(s). In certain embodiments,the compound comprises at least about 90% by weight of the designatedenantiomer and at most about 10% by weight of the other enantiomer orother stereoisomer(s). In certain embodiments, the compound comprises atleast about 95% by weight of the designated enantiomer and at most about5% by weight of the other enantiomer or other stereoisomer(s). Incertain embodiments, the compound comprises at least about 96.6% byweight of the designated enantiomer and at most about 3.4% by weight ofthe other enantiomer or other stereoisomer(s). In certain embodiments,the compound comprises at least about 97% by weight of the designatedenantiomer and at most about 3% by weight of the other enantiomer orother stereoisomer(s). In certain embodiments, the compound comprises atleast about 99% by weight of the designated enantiomer and at most about1% by weight of the other enantiomer or other stereoisomer(s). Incertain embodiments, the compound comprises at least about 99.9% byweight of the designated enantiomer and at most about 0.1% by weight ofthe other enantiomer or other stereoisomer(s). In certain embodiments,the weights are based upon total weight of the compound.

Another aspect provides for salts, including pharmaceutically acceptablesalts, of compounds of Formula I or II and pharmaceutical compositionscomprising a pharmaceutically acceptable salt of compounds of Formula Ior II. Salts of compounds of Formula I or II include an inorganic baseaddition salt such as sodium, potassium, lithium, calcium, magnesium,ammonium, aluminum salts or organic base addition salts.

Another aspect provides for anhydrates, hydrates and solvates ofcompounds of Formula I or II and pharmaceutical compositions comprisinga pharmaceutically acceptable anhydrates, hydrates and solvates ofcompounds of Formula I or II. Included are an anhydrate, hydrate orsolvate of a free form or salt of a compound of Formula I or II.Hydrates include, for example, a hemihydrate, monohydrate, dihydrate,trihydrate, quadrahydrate, pentahydrate, sesquihydrate.

In certain embodiments, the compounds of Formula I or II are able todisplace radiolabeled glucagon from the human glucagon receptor by atleast 15% at 1000 nM. In one embodiment, the compounds of Formula I orII are able to displace at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, or 99% of radiolabeled glucagon from the humanglucagon receptor as described in Example A.

Alternatively, the activities of the compounds of Formula I or II can bedescribed in terms of the concentrations of compounds required fordisplacement of 50% of the radiolabeled glucagon from the human glucagonreceptor (the IC₅₀ values) according to the methods of Example A. In oneembodiment, the IC₅₀ values for the compounds of Formula I are less than<10,000 nM, 9,000 nM, 8,000 nM, 7,000 nM, 6,000 nM, 5,000 nM, 4,000 nM,3,000 nM, 2,000 nM, 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM,400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM or 5 nM.

In another alternative, the activities of the compounds of Formula I orII can be described in terms of the concentrations of compounds requiredfor functional antagonism of glucagon in hepatocytes from variousspecies. The EC₅₀ is determined using the method of Example B. In oneembodiment, the EC₅₀ values for the compounds of Formula I or II areless than <10,000 nM, 9,000 nM, 8,000 nM, 7,000 nM, 6,000 nM, 5,000 nM,4,000 nM, 3,000 nM, 2,000 nM, 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM,500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM or 5 nM.

The compounds of Formula I or II disclosed herein also exhibit theability to reduce blood glucose in an animal. In certain aspects,circulating blood glucose in fasting or non-fasting (freely-feeding)animals can be reduced between 10% and 100%. A reduction of 100% refersto complete normalization of blood glucose levels, not 0% blood glucoselevels. Normal blood glucose in rats, for example, is approximately 80mg/dl (fasted) and approximately 120 mg/dl (fed). Thus, contemplatedherein is a method for reducing excessive circulating blood glucoselevels in fasting or freely fed animals (e.g. rat), by administered 10mg/kg of a compound of Formula I, by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 99%.

c. Administration

Provided herein are pharmaceutical compositions including a compoundprovided herein as an active ingredient, e.g., a compound of Formula Ior II, or a pharmaceutically acceptable salt, solvate, or prodrugthereof; in combination with a pharmaceutically acceptable vehicle,carrier, diluent, excipient, or a mixture thereof.

The pharmaceutical compositions may be formulated in various dosageforms, including, but limited to, the dosage forms for oral, parenteral,subcutaneous, intramuscular, transmucosal inhaled, ortopical/transdermal administration. The pharmaceutical compositions mayalso be formulated as modified release dosage forms, including, but notlimited to, delayed-, extended-, prolonged-, sustained-, pulsatile-,controlled-, accelerated- and fast-, targeted-, programmed-release, andgastric retention dosage forms. These dosage forms can be preparedaccording to conventional methods and techniques known to those skilledin the art (see, Remington: The Science and Practice of Pharmacy, supra;Modified-Release Drug Deliver Technology, Rathbone et al., Eds., Drugsand the Pharmaceutical Science, Marcel Dekker, Inc.; New York, N.Y.,2003; Vol. 126).

The pharmaceutical compositions provided herein may be provided in aunit- or multiple-dosage form. A unit-dosage form, as used herein,refers to a physically discrete unit suitable for administration to asubject as is known in the art. Examples of a unit-dosage form includean ampoule, syringe, and individually packaged tablet and capsule. Aunit-dosage form may be administered in fractions or multiples thereof.

The pharmaceutical compositions provided herein may be administered atonce, or multiple times at intervals of time. It is understood that theprecise dosage and duration of treatment may vary with the age, weight,and condition of the patient being treated, and may be determinedempirically using known testing protocols or by extrapolation from invivo or in vitro test or diagnostic data. It is further understood thatfor any particular individual, specific dosage regimens can be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thepharmaceutical compositions provided herein.

Exemplary pharmaceutical compositions and components for use therewithare described in U.S. Provisional Application No. 61/088,697, thecontents of which are herein incorporated by reference.

d. Methods of Use

In one embodiment, provided herein is a method of treating, preventing,or ameliorating one or more symptoms of a condition, disorder, ordisease associated with impaired glucose tolerance, a metabolicsyndrome, or a glucagon receptor, comprising administering to a subjecthaving or being suspected to have such a condition, disorder, ordisease, a therapeutically effective amount of a compound providedherein, e.g., a compound of Formula I or II, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof; or a pharmaceuticalcomposition thereof. In one embodiment, the subject is a mammal. Inanother embodiment, the subject is a human.

In another embodiment, provided herein is a method of treating,preventing, or ameliorating one or more symptoms of a condition,disorder, or disease responsive to a decrease in the hepatic glucoseproduction or in the blood glucose level of a subject, comprisingadministering to the subject having or being suspected to have such acondition, disorder, or disease, a therapeutically effective amount of acompound provided herein, e.g., a compound of Formula I or II, or apharmaceutically acceptable salt, solvate, or prodrug thereof; or apharmaceutical composition thereof. In one embodiment, the subject is amammal. In another embodiment, the subject is a human.

The conditions and diseases treatable with the methods provided hereininclude, but are not limited to, type 1 diabetes, type 2 diabetes,gestational diabetes, ketoacidosis, nonketotic hyperosmolar coma(nonketotic hyperglycemia), impaired glucose tolerance (IGT), insulinresistance syndromes, syndrome X, low HDL levels, high LDL levels,hyperglycemia, hyperinsulinemia, hyperlipidemia, hypertriglyceridemia,hyperlipoproteinemia, hypercholesterolemia, dyslipidemia,arteriosclerosis, atherosclerosis, glucagonomas, acute pancreatitis,cardiovascular diseases, hypertension, cardiac hypertrophy,gastrointestinal disorders, obesity, vascular resenosis, pancreatitis,neurodegenerative disease, retinopathy, nephropathy, neuropathy,accelerated gluconeogenesis, excessive (greater than normal levels)hepatic glucose output, and lipid disorders.

Provided herein are also methods of delaying the time to onset orreducing the risk of the development or progression of a disease orcondition responsive to decreased hepatic glucose production orresponsive to lowered blood glucose levels.

Depending on the condition, disorder, or disease to be treated and thesubject's condition, a compound provided herein may be administered byoral, parenteral (e.g., intramuscular, intraperitoneal, intravenous orintraarterial (e.g., via catheter). ICV, intracistemal injection orinfusion, subcutaneous injection, or implant), inhalation, nasal,vaginal, rectal, sublingual, and/or topical (e.g., transdermal or local)routes of administration, and may be formulated alone or together insuitable dosage unit with a pharmaceutically acceptable vehicle,carrier, diluent, excipient, or a mixture thereof, appropriate for eachroute of administration.

The dose may be in the form of one, two, three, four, five, six, or moresub-doses that are administered at appropriate intervals per day. Thedose or sub-doses can be administered in the form of dosage unitscontaining from about from about 0.01 to 2500 mg, from 0.1 mg to about1,000 mg, from about 1 mg to about 1000 mg, from about 1 mg to about 500mg, from about 0.1 mg to about 500 mg, from about 0.1 mg to about 100mg, from about 0.5 mg about to about 100 mg, from about 1 mg to about100 mg, from about 10 mg to about 1000 mg, from about 10 mg to about 500mg, or from about 10 mg to about 100 mg of active ingredient(s) perdosage unit. For example, the dose or subdoses can be administered inthe form of dosage units containing about 10 mg, about 25 mg, about 50mg, about 75 mg, about 100 mg, about 250 mg, about 300 mg, about 400 mg,about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, orabout 1000 mg. If the condition of the patient requires, the dose can,by way of alternative, be administered as a continuous infusion.

In certain embodiments, an appropriate dosage level is about 0.01 toabout 100 mg per kg patient body weight per day (mg/kg per day), about0.01 to about 50 mg/kg per day, about 0.01 to about 25 mg/kg per day, orabout 0.05 to about 10 mg/kg per day, which may be administered insingle or multiple doses. A suitable dosage level may be about 0.01 toabout 100 mg/kg per day, about 0.05 to about 50 mg/kg per day, or about0.1 to about 10 mg/kg per day. Within this range, the dosage may beabout 0.01 to about 0.1, about 0.1 to about 1.0, about 1.0 to about 10,or about 10 to about 50 mg/kg per day.

For oral administration, the pharmaceutical compositions can be providedin the form of tablets containing 1.0 to 1,000 mg of the activeingredient, particularly about 1, about 5, about 10, about 15, about 20,about 25, about 50, about 75, about 100, about 150, about 200, about250, about 300, about 400, about 500, about 600, about 750, about 800,about 900, and about 1,000 mg of the active ingredient for thesymptomatic adjustment of the dosage to the patient to be treated. Thecompositions may be administered on a regimen of 1 to 4 times per day,including once, twice, three times, and four times per day. In variousembodiments, the compositions may be administered before a meal, after ameal, in the morning hours, after awakening, in the evening hours,and/or at bedtime.

It will be understood, however, that the specific dose level, frequency,and timing of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the host undergoing therapy.

In still another embodiment, provided herein is a method of modulatingthe biological activity of a glucagon receptor, comprising contactingthe receptor with one or more of the compounds provided herein, e.g., acompound of Formulas I or II, including a single enantiomer, a mixtureof enantiomers, or a mixture of diastereomers thereof, or apharmaceutically acceptable salt, solvate, or prodrug thereof; or apharmaceutical composition thereof. En one embodiment, the glucagonreceptor is expressed by a cell.

The compounds provided herein may also be combined or used incombination with other therapeutic agents useful in the treatment,prevention, or amelioration of one or more symptoms of the conditions,disorders, or diseases for which the compounds provided herein areuseful. As used herein, the term “in combination” includes the use ofmore than one therapeutic agents. However, the use of the term “incombination” does not restrict the order in which therapeutic agents areadministered to a subject with a condition, disorder, or disorder. Afirst therapeutic agent (e.g., a therapeutic agent such as a compoundprovided herein) can be administered prior to (e.g., 5 min, 15 min, 30min, 45 min, 1 hr, 2 hrs, 4 hrs, 6 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs,96 hrs, 1 wk, 2 wks, 3 wks, 4 wks, 5 wks, 6 wks, 8 wks, or 12 wksbefore), concomitantly with, or subsequent to (e.g., 5 min, 15 min, 30min, 45 min, 1 hr, 2 hrs, 4 hrs, 6 hrs, 12 hrs, 24 hrs. 48 hrs, 72 hrs,96 hrs, 1 wk, 2 wks, 3 wks, 4 wks, 5 wks, 6 wks, 8 wks, or 12 wks after)the administration of a second therapeutic agent to a subject to betreated.

When a compound provided herein is used contemporaneously with one ormore therapeutic agents, a pharmaceutical composition containing suchother agents in addition to the compound provided herein may beutilized, but is not required. Accordingly, the pharmaceuticalcompositions provided herein include those that also contain one or moreother therapeutic agents, in addition to a compound provided herein.

In one embodiment, the other therapeutic agent is an antidiabetic agent.Suitable antidiabetic agents include, but are not limited to, insulinsensitizers, biguanides (e.g., buformin, metformin, and phenformin),PPAR agonists (e.g., troglitazone, pioglitazone, and rosiglitazone),insulin and insulin mimetics, somatostatin, α-glucosidase inhibitors(e.g., voglibose, miglitol, and acarbose), dipeptidyl peptidase-4inhibitors, liver X receptor modulators, insulin secretagogues (e.g.,acetohexamide, carbutamide, chlorpropamide, glibornuride, gliclazide,glimerpiride, glipizide, gliquidine, glisoxepid, glyburide, glyhexamide,glypinamide, phenbutamide, sulfonylureas, tolazamide, tolbutamide,tolcyclamide, nateglinide, and repaglinide), other glucagon receptorantagonists, GLP-1, GLP-1 mimetics (e.g., exenatide, liraglutide, DPPIVinhibitors), GLP-1 receptor agonists, GIP, GIP mimetics, GIP receptoragonists, PACAP, PACAP mimetics, PACAP receptor 3 agonists, cholesterollowering agents, HMG-CoA reductase inhibitors (e.g., statins, such aslovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin,itavastatin, rivastatin, NK-104 (a.k.a. itavastatin, nisvastatin, andnisbastatin), and ZD-4522 (also known as rosuvastatin, atavastatin, andvisastatin)), a cholesterol absorption inhibitor (e.g., ezetimibe),sequestrants, nicotinyl alcohol, nicotinic acid and salts thereof, PPARα agonists, PPAR α/γ dual agonists, inhibitors of cholesterolabsorption, acyl CoA:cholesterol acyltransferase inhibitors,anti-oxidants, PPAR δ agonists, antiobesity compounds, ileal bile acidtransporter inhibitors, anti-inflammatory agents, and protein tyrosinephosphatase-1B (PIP-1B) inhibitors.

The dosages given will depend on absorption, inactivation and excretionrates of the drug as well as other factors known to those of skill inthe art. It is to be noted that dosage values will also vary with theseverity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimens andschedules should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions.

The weight ratio of a compound provided herein to the second activeingredient depends upon the effective dose of each ingredient.Generally, an effective dose of each will be used. Thus, for example,when a compound provided herein is combined with a PPAR agonist theweight ratio of the compound provided herein to the PPAR agonist willgenerally range from about 1000:1 to about 1:1000 or about 200:1 toabout 1:200. Combinations of a compound provided herein and other activeingredients will generally also be within the aforementioned range, butin each case, an effective dose of each active ingredient should beused.

Synthesis of Compounds

Compounds of Formula I and II can be prepared according to themethodology outlined in the following general synthetic schemes or withmodifications of these schemes that will be evident to persons skilledin the art, or by other methods readily known to those of skill in theart.

In the following sections, the following abbreviations have thefollowing meanings: THF: Tetrahydrofuran; DME: 1,2-Dimethoxyethane; DMF:N,N-Dimethylformamide; DCC: N,N′-Dicyclohexylcarbodiimide; EDCI or EDC:1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride; LiHMDS:Lithium hexamethyldisilyl azide; HOBt: 1-Hydroxybenzotriazole; EtOAc:Ethyl acetate; EtOH: Ethanol; IPA: iso-Propanol; ACN: Acetonitrile;DIPEA: N,N-Diisopropyl-ethyl amine; and MTBE: Methyl-tert-butyl ether.

a. Synthesis of Various Building Blocks:

The carboxylic acids 3 can be generated using standard methods. As shownin Scheme 1, a carboxylic ester or acid 1 can be alkylated by reactionwith a base (such as lithium diisopropylamide or lithiumhexamethyldisilylamide) in a suitable solvent (such as THF or DME)followed by reaction with an aralkyl halide to generate intermediates 2.In one embodiment, when Ra is not hydrogen, then the Ra and Rb groupsare adequately chosen so that liberation of the carboxylic acid togenerate 3 can take place selectively when Ra is H, 2 and 3 representthe same intermediate). For example, if Ra is a methyl or ethyl group,an Rb group can be a benzyl, t-butyl, 2-trimethylsilylethyl group orother groups that can be selectively removed under conditions where theester group Ra would remain intact such as hydrogenolysis for the benzylgroup, mild acid such as trifluoroacetic acid for the t-butyl group or afluoride source for the 2-trimethylsilylethyl group such as a tetraalkylammonium fluoride (e.g. tetrabutyl ammonium fluoride).

An alternative route for the synthesis of this particular buildingblock, shown in Scheme 2, involves the condensation of an acetic acidderivative 1 with an aldehyde or a ketone leading to the α,β-unsaturatedester intermediate 4. The esters 4 can be hydrogenated under conditionsthat are well-documented in the literature (for example, hydrogenatmosphere and palladium on carbon as a catalyst in a solvent such asethanol) to generate the carboxylate esters 3.

Alternatively, if R⁴⁴ in 4 is H (compound 5), 1,4-addition of an alkylgroup can take place by reaction with a suitable carbon nucleophile(e.g. copper mediated reaction of alkyl lithium or alkyl Grignardreagents) to yield compounds 3 where R⁴⁴ is alkyl (Scheme 3).

Scheme 4 shows an alternative route to precursors 5, involving thepalladium catalyzed reaction of a vinylic halide 7 with anorganometallic reagent such as an aryl boronic acid or an aryl stannane.These vinylic halides 7 (where Hal represents bromide or iodide), can begenerated from the corresponding benzaldehydes and a halogenatedHorner-Emmons reagent (RO)₂P(O)CH(Hal)CO₂Ra (Toke et al, Tetrahedron 51,9167 (1995); Vanderwal et al, J. Am. Chem. Soc., 125 (18), 5393-5407(2003)) in the presence of base or by the reaction of the same startingaldehyde with [Ph₃P=C(IPh)CO₂Ra]⁽⁺⁾[BF₄]⁽⁻⁾ in dichloromethane in thepresence of a halide source such as tetra-n-butyl ammonium bromide ortetra-n-butyl ammonium iodide (Huang et al, J. Org. Chem 67, 8261(2002))

It is recognized that the carbon atom alpha to the central carbonylgroup is an asymmetric center. The synthesis of compounds providedherein in enantiomerically pure form can be achieved by utilization ofthe methods described above if the starting material 8 exists inenantiomerically pure form. An optically pure precursor 8* or 8**, canbe generated by resolution of racemic 8 or by use of synthetic methodsthat generate the asymmetric center in an enantioselective manner.

Resolution methods include the generation of a diastereomeric mixture ofcarboxylate salts with an optically active amine, which may be separatedby fractional crystallization. Acidification of the individualdiastereomeric salts and isolation of the carboxylic acid affords theindividual enantiomers of the carboxylic acid (D. Kozma: ‘CRC Handbookof Optical Resolutions via Diastereomeric Salt Formation’ CRC Press,2001). Alternatively, diastereomeric mixtures of ester or amidederivatives may be prepared by condensation of the racemic carboxylicacid with an optically active alcohol or amine, respectively; thesediastereomers may be separated by chromatographic methods and/orfractional crystallization. The pure enantiomers are then generated fromthe individual diastereomers by reconversion to the carboxylic acid,using methods that are well established in the literature (Scheme 5).

Methods that generate the chiral center in an enantioselective mannerinclude, but are not limited to, the alkylation of precursors containinga chiral auxiliary Xc. This may generate two diastereomers, which may beseparated by fractional crystallization or chromatography (Scheme 6).After the separation of the diastereomers, they can be converted intothe enantiomerically pure acid 3 and its enantiomer 3* by knownprocedures and further elaborated into the compounds provided herein asdescribed in the Examples below.

Asymmetric centers may be present in other positions of the molecule. Asan example, substitution on a cyclohexenyl group generates a new chiralcenter in the compound of Example 1. This center can be established inan appropriately functionalized precursor prior to construction of thetarget molecule. A potential route to this chiral precursor involves thedesymmetrization of a racemic ketone as illustrated in Scheme 7. Thereaction of 4-t-butylcyclohexanone with a chiral amide base has beenreported to generate the corresponding chiral enolate in anenantioselective manner [Busch-Petersen and Corey. Tetrahedron Letters41, 6941(2000), Lyapkalo et al, Synlett 1292(2001)]. Conversion of theenolate into a trifluoromethanesulfonate or a nonafluorobutanesulfonate[Busch-Petersen and Corey, Tetrahedron Letters 41, 6941(2000), Lyapkaloet al. Synlett 1292(2001)], leads to a chiral precursor that may be usedin subsequent steps (A specific enantiomer is shown below, but it shouldbe understood that either enantiomer can be synthesized by modificationsof this method). The precursor 9 so obtained can then be elaborated intothe single enantiomer as described above.

In a related manner, the two enantiomers of a 4-substitutedcyclohex-1-enyl system can be obtained through resolution of thecorresponding racemic alkene. For example, (enantiospecific orenantioselective) reaction of an alkene 10 (wherein R⁵⁰ and R⁵¹ aredifferent groups) generates a mixture of diastereomers which uponseparation provides 11 and 12. Regeneration of the alkene provides thetwo enantiomers 13 and 13* (Scheme 8).

In addition to the methods described above, optically pure compounds canbe obtained from their racemic parent compounds through chromatographicmethods that utilize a chiral stationary phase.

A method that can be used to synthesize compounds of formula I isexemplified below (Scheme 9). The carboxylic acids 8 are converted tothe corresponding amides by methods known for amide bond formationreactions. As an example, generation of an acid chloride 14 from 8 takesplace under standard conditions (e.g. thionyl chloride in toluene oroxalyl chloride and catalytic DMF in dichloromethane). Treatment of acidchloride 14 with amities or anilines generates the amides 15.Alternatively, amines can be directly coupled with the carboxylic acid 8by use of an activating agent (for example, DCC or EDCI with or withouta catalyst such as DMAP or HOBT) to directly generate the amides 15.When L is a halo group such as bromo or iodo, aryl amides 15 can befurther functionalized through metal-mediated (e.g. Palladium) C—C bondcoupling reactions to give further L-functionalized amides 15.Hydrolysis of the ester group of 15 (e.g. Rb=—CH₃ or —C(CH₃)₃) resultsin a carboxylic acid 15 (wherein Rb=H), which can then be coupled withtaurine derivatives using standard amide bond forming reactions togenerate the targeted compounds 16.

The amide bond in the last step can also be formed by other reportedmethods known for amide bond formation, for example, reaction of anN-hydroxysuccinimidyl ester of 15 (Rb=O-succinimidyl) and taurine givesthe target taurine amide derivative 16. Other activated esters (e.g.pentafluorophenyl esters) can also be used to effect the amide bondformation.

The following examples are provided so that this disclosure can be morefully understood. They should not be construed as limiting thedisclosure in any way.

EXAMPLES Biological Examples Example A—Human Glucagon Receptor Affinity

Compounds provided herein are dissolved in a suitable solvent (e.g.,dimethylsulfoxide) at a concentration of 10 mM and then diluted inbuffer (e.g., 50 mM Hepes, pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, and 0.2% BSA)to concentrations ranging from 1 nM to 100 μM. Compounds (20 μL/well)and [¹²⁵I]glucagon at the final concentration of 0.125 nM (20 μl/well)(Perkin Elmer) are added to and mixed in wells of a 96-well plate(Costar, Corning) containing 120 μL of buffer. Next, an appropriatealiquot of a membrane preparation containing the human glucagon receptor(isolated from human liver samples or obtained from a recombinant cellline) is added to the wells. The binding mixtures are incubated for 2hrs at room temperature. In the meantime, a MultiScreen 96-well filterplate (Millipore) is treated with 200 μL of the buffer, which isvacuumed through the filter just before the binding mixtures aretransferred to the plate. At the end of incubation, binding mixtures aretransferred to the wells of the MultiScreen 96-well filter plate andfiltered through by applying vacuum. The plate is washed once with 200μL per well of the buffer, and the filters are dried and counted using agamma counter.

Compounds provided herein have been shown to have high affinity for theglucagon receptor. Examples of some compounds are provided in the Tablebelow. The Table below displays the results of testing the compoundsshown in the human glucagon receptor binding assay. Also shown are datafrom the human hepatocyte assay, oral bioavailability in the rat, andglucose lowering in the db/db mouse (see Examples below for assaydescriptions). The column marked “stereo” indicates whether the testedcompound tested was racemic or the R-isomer (rac=racemic).

          stereo       Hu IC50 (nM)       Hu Hepatocyte EC50 (nM)        OBAV in rat     % Glu lowering in db/db mouse @ 30 mg/kg

R 5 4 73 57

R 10 12 52 45

R 9 3 54 53

rac 18 27 — —

R 6 31 62 33

R 8 36 69 29

rac 18 412 — —

rac 10 194 — 45

rac 12 41 — 58

rac 15 132 — —

rac 15 329 — —

rac 11 560 — —

rac 15 215 — —

rac 11 30 — 20

rac 19 — — —

rac 18 60 — —

rac 12 661 — —

rac 10 — — —

rac 16 — — —

rac 8 116 — —

rac 18 161 — —

rac 12 30 — —

rac 17 49 48 42

rac 16 — — —

rac 16 78 — —

rac 15 137 — —

rac 20 143 — —

rac 13 62 — 39

rac 9 26 38 56

rac 14 32 — —

rac 19 108 — —

rac 15 30 — 28

rac 18 61 — —

rac 12 241 — —

rac 8 167 — —

rac 12 — — —

rac 18 — — —

rac 16 — — 25

rac 12 — —

rac 18 — — —

rac 10 — — —

rac 14 — — —

rac 10 192 — —

rac 17 65 — 30

rac 10 — — —

The compounds of Formula I provided herein which have been testeddisplace radiolabeled glucagon from the human glucagon receptor with anIC₅₀ of <15 nM.

For certain compounds, the R-enantiomer displays up to 5-fold higheraffinity for the human glucagon receptor than the S-enantiomer.

The Table below displays the relative potency in the human glucagonreceptor binding assay of the R- vs. S-isomers of the compounds shown.(The stereocenter being changed is marked with an asterisk; the R-isomeris shown in the drawing.)

      Hu IC50    (nM)    R   S

14 61

 6 20

 7 19

11 37

10 43

10 49

The compounds in the above table display nanomolar affinity for thehuman glucagon receptor. For certain compounds, the R-enantiomerdisplays up to 5-fold higher affinity for the human glucagon receptorthan the S-enantiomer.

Example B—Functional Antagonism in Hepatocytes from Various Species

Primary human, monkey, dog, rat, or mouse hepatocytes are seeded ontocollagen-coated 24-well plates in Williams E medium (supplemented with10% fetal bovine serum) and incubated at 37° C. overnight in M199 medium(supplemented with 15 mM glucose and 10 nM human insulin). The followingday cells are washed twice with a glucose-free Kreb-bicarbonate buffer,pH 7.4, containing 0.1% BSA. Cells are then incubated at 37° C. with theaforementioned buffer containing 1 nM glucagon and varyingconcentrations of a glucagon antagonist (0-100 microM). Control wellswithout glucagon or antagonist are also included. After 1 hour, analiquot of the medium is removed and analyzed for glucose content bymeans of the glucose oxidase method. The background glucose levelsobserved in the control wells are subtracted from the glucagon andantagonist containing wells. A graph of % glucose concentration vs drugconcentration is plotted and an EC50 value for inhibition of glucoseproduction generated using Sigmaplot software (SAS, Cary, N.C.).Alternatively, intracellular cAMP levels are measured using standardkits and EC50 values determined by plotting these levels against drugconcentration. Antagonists of the glucagon receptor inhibitglucagon-induced cAMP production.

The R-enantiomer compounds of Formula I provided herein which have beentested show functional antagonism of glucose production in humanhepatocytes with an EC₅₀ of <40 nM.

The compounds disclosed herein display significant functional antagonismof glucose production in human hepatocytes. For certain compounds, theR-enantiomer display's up to 50-fold greater functional antagonism inhuman hepatocytes than the S-enantiomer.

The Table below displays the relative potency in the human hepatocytefunctional assay of the R- vs. S-isomers of the compounds shown. (Thestereocenter being changed is marked with an asterisk; the R-isomer isshown in the drawing.)

      Hu cell EC50 (nM)   R   S  

37 1177 

 7 350

 6 165

33 213

Example C—Glucose Lowering in Diabetic Animals

The effects of compounds provided herein on blood glucose levels areassessed in animal models of type 1 or 2 diabetes such as, but notlimited to, the db/db mouse, the Zucker fatty (ZF) rat, the Zuckerdiabetic (ZDF) rat, the glucagon-challenged dog, the alloxan- orstreptozotocin-treated mouse or rat, the NOD mouse or the BB rat.

Compounds are dissolved in an appropriate vehicle such as polyethyleneglycol-400 or cyclodextrin and administered to animals at doses of 0.1to 100 mg/kg either by intraperitoneal injection, intravenous injection,or oral gavage. Animal models used in this evaluation [e.g., the db/dbmouse, the ZF rat, the ZDF rat, the glucagon-challenged (0.3-5 μg/kg)dog, the alloxan- or streptozotocin-treated mouse or rat, the NOD mouse,or BB rat] are either freely-feeding or fasted from 3 to 24 hours priorto compound administration. In some instances, animals may be subjectedto a glucose tolerance test following compound administration byintravenous or oral administration of up to 2 g/kg of glucose. Bloodglucose levels are assessed in blood samples obtained by tail bleed orby sampling an appropriate blood vessel by means of a syringe orcatheter. Glucose is measured using a portable glucometer such as theOneTouch or RemoCue meters at regular time intervals for 0.10 up to 24hours. The extent of blood glucose lowering elicited by the compounds ofFormula I or II is determined by comparison to those in control animalsadministered only the vehicle. The percentage of blood glucose loweringattained is calculated relative to blood glucose levels invehicle-treated nondiabetic or non-glucagon-challenged control animals.

Example D—Glucose Lowering in Db/Db Mice

To assess the effects of compounds provided herein on blood glucoselevels in the db/db mouse, an animal model of type 2 diabetes, compoundsare dissolved in polyethylene glycol-400 and administered by oral gavageto db/db mice in the freely-feeding state at doses of 30 and/or 100mg/kg. Blood glucose levels are assessed in blood samples obtained bytail bleed at baseline (prior to drug administration) and at regulartime intervals over 24 hrs using a portable glucometer such as theOneTouch or HemoCue meters. The magnitude of blood glucose loweringelicited by the compounds provided herein is determined by comparison tothose in db/db mice administered only the vehicle. The percentageglucose lowering is calculated by factoring in the blood glucose levelsof vehicle-treated lean db/+ (heterozygote) mice, with 100% representingthe normalization of blood glucose levels from the hyperglycemic state(vehicle-treated db/db mice) to the normoglycemic state (vehicle-treateddb/+ mice).

The compounds disclosed herein which have been tested lowered bloodglucose of db/db mice in the freely-feeding state. In particular, thepercentage blood glucose lowering achieved ranged from 36 to 57%relative to lean control animals.

The compounds disclosed herein have pronounced antihyperglycemicactivity in animal models of type 2 diabetes.

EXAMPLES Chemical Synthetic Examples Example 1: Sodium;2-{4-[2-[4-(4-tert-butyl-cyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonateStep 1: 4-[2-(4-Bromophenyl)-2-carboxy-ethyl]-benzoic acid methyl ester

In a 3-neck flask, a solution of 4-bromophenyl acetic acid (26.91 g) inTHF (485 mL) was cooled to <10° C. under a nitrogen atmosphere. Asolution of LiHMDS in THF (263 mL, 1.0 M) was added dropwise, ensuringthat the internal temperature remained at <10° C. After the addition wascomplete, the mixture was stirred at 0° C. for about 15 min. The coolingbath was the removed and the reaction mixture was allowed to warm up to20° C.

The reaction mixture was then cooled to <−60° C. From an additionfunnel, a solution of 4-bromomethyl methylbenzoate (29.53 g) in THF (270mL) was added dropwise, ensuring that the temperature did not rise above−60° C. After the addition was complete, the mixture was stirred at −60°C. for about 15 min, and poured over 300 mL of cold 1M aqueous HCl(saturated with sodium chloride). The organic layer was washed with 1Maqueous HCl (saturated with sodium chloride). The combined aqueouslayers were extracted with toluene (50 mL). The combined organic phaseswere then dried over magnesium sulfate and concentrated under reducedpressure. The crude residue was recrystallized from toluene to yield thecarboxylic acid as a white solid. HNMR (300 MHz, DMSO-d₆) 12.54 (1H,broad s), 7.82 (2H, d, J=6.4 Hz), 7.49 (2H, d, J=6.7 Hz), 7.32 (2H, d,J=8, 2 Hz), 7.26 (2H, d, J=8.5 Hz), 3.95 (1H, t, J=7.9 Hz), 3.81 (3H,s), 3.3 (1H, m, overlaps with residual HOD), 3.03 (1H, m)

Step 2:4-{2-(4-Bromo-phenylcarbamoyl)-2-[4-(4-tert-butyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoicacid methyl ester

To a solution of 4-[2-(4-Bromophenyl)-2-carboxy-ethyl]-benzoic acidmethyl ester (step 1 above, 0.6 g) in THF: ethanol: water (6 mL: 3 mL:1.5 mL) added 4-t-butyl-1-cyclohexenyl-boronic acid (0.5 g),PdCl₂(P(o-tolyl)₃)₂, and sodium carbonate (0.7 g). The resulting mixturewas heated at 125° C. for a 1 h period. The reaction was then cooled toroom temperature, treated with an excess of aqueous HCl (1M) and theresulting heterogeneous mixture was filtered through a celite pad. Theorganic phase was dried over sodium sulfate and concentrated underreduced pressure. The crude residue was dissolved in toluene (25 mL),treated with thionyl chloride (0.26 mL) and heated at 100° C. for a 1 hperiod. The toluene was removed under reduced pressure. The resultingacid chloride was redissolved in toluene (15 mL), treated with4-bromoaniline (0.3 g) and diisopropyl ethyl amine (0.3 mL), and heatedat 100° C. for a 1 h period. After cooling to room temperature, themixture was partitioned between ethyl acetate and 1M aqueous HCl: Theorganic layer was washed (water, saturated sodium chloride), dried overmagnesium sulfate and concentrated under reduced pressure. The productobtained was carried to the next step without further purification.

Step 3: Sodium;2-(4-{2-(4-bromo-phenylcarbamoyl)-2-[4-(4-tert-butyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoylamino)-ethanesulfonate

A solution of4-{2-(4-Bromo-phenylcarbamoyl)-2-[4-(4-tert-butyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoicacid methyl ester (Step 2, 0.8 g) in THF: methanol: water (8 mL: 6 mL: 2mL) was treated with lithium hydroxide (0.4 g) and stirred at roomtemperature for 16 h. Added an excess of aqueous HCl (1M) and extractedwith ethyl acetate. The organic phase was washed with saturated sodiumchloride in water and dried over sodium sulfate. The solvents were thenremoved under reduced pressure. The residue obtained was dissolved inDMF (0.10 mL), and treated with EDCI (0.4 g). HOBt-H₂O (0.32 g), taurine(0.26 g) and diisopropyl ethyl amine (0.71 mL). The reaction mixture wasthen stirred at room temperature for a 16 h period. The solvent wasremoved under reduced pressure. The residue was partitioned betweenethyl acetate and 1M aqueous HCl. The organic phase was washed withsaturated sodium chloride, and concentrated. The residue in methanol wastreated with an excess of sodium hydroxide and loaded on top of a C-18reverse phase flash chromatography column. The column was eluted with anacetonitrile—water gradient to afford the sodium salt of the sulfonateas a white solid. LCMS m/z: 665.6 [C₃₄H₃₈N₂O₅BrS]⁻

Step 4

To a solution sodium;2-(4-{2-(4-bromo-phenylcarbamoyl)-2-[4-(4-tert-butyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoylamino)-ethanesulfonate(Step 3 above, 96 mg) in DME: ethanol: water (2 mL: 1 mL: 0.5 mL) added4-t-butyl-1-cyclohexenyl-boronic acid (0.5 g), PdCl₂(P(o-tolyl)₃))₂, andsodium carbonate (0.7 g). The resulting mixture was heated at 125° C.for a 1 h period. The reaction was then cooled to room temperature,treated with an excess of aqueous HCl (1M) and the resultingheterogeneous mixture was filtered through a celite pad. The mixture waspartitioned (ethyl acetate/water). The organic phase was washed withsaturated sodium chloride, and concentrated. The residue in methanol wastreated with an excess of sodium hydroxide and loaded on top of a C-18reverse phase flash chromatography column. The column was eluted with anacetonitrile—water gradient to afford the sodium salt of the sulconateas a white solid. LC-MS m/z 711.6 [C₄₄H₄₄N₂O₅ClS]⁻

Example 2:Sodium-2-(4-{2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-2-[3-(4,4-dimethyl-cyclohexyl)-phenyl]-ethyl}-benzoylamino-ethanesulfonic acid Step 1:4-Benzyl-3-[2-(3-bromo-phenyl)-acetyl]-oxozolidin-2-one

To a solution of (3-bromo-phenyl)-acetic acid (5.0 g, 23.2 mmol) inCH₂Cl₂ (50 mL) at room temperature was added oxalyl chloride (5.86 g,46.5 mmol). The reaction mixture was stirred at room temperature forovernight and the solvent was removed under reduced pressure. Theresidue was dried under vacuum for 3-4 h and used without furtherpurification.

In a separate flask, to a stirred solution ofR-(±)-4-benzyl-oxazolidinone (4.34 g, 24.5 mmol) in THF (30 mL) at −78°C. was added n-BuLi (26.7 mL, 26.7 mmol, 1.0 M solution in hexane). Thereaction mixture was stirred for 1 h, at −78° C., then the crude acidchloride (5.2 g, 22.3 mmol) in THF was added dropwise. The mixture wasstirred for 2 h at −78° C. and allowed to warm to rt and stirred foranother hour (monitored by TLC). The reaction was quenched withsaturated NH₄Cl solution (100 mL) and stirred for 10 min. The reactionmixture was extracted with ethyl acetate (2×250 mL) and the combinedorganic layers were washed with brine, dried over MgSO₄ and concentratedunder reduced pressure. The crude product was purified by columnchromatography on silica gel, eluting with EtOAc-hexanes (5-25%) toafford 4-benzyl-3-[2-(3-bromo-phenyl)-acetyl]-oxozolidin-2-one. ¹H NMR(300 MHz, CDCl₃): 7.51 (d, J=2.1 Hz, 1H), 7.44 (dd, J=3.9, 4.8 Hz, 1H),7.32-7.20 (m, 6H), 7.15 (d, J=3.9 Hz, 1H), 4.60 (m, 1H), 4.30 (m, 2H),4.26-4.19 (m, 3H), 3.27 (dd, J=1.8, 8.1 Hz, 1H), 2.78 (dd, J=5.4, 7.8Hz, 1H); TLC conditions: Uniplate silica gel, 250 microns; mobilephase=ethyl acetate/hexanes (5:1); R_(f)=0.6.

Step 2:4-{3-(4-Benzyl-2-oxo-oxazolidin-3-yl)-2-[4-(3-bromophenyl)-3-oxo-propyl}-benzoicacid tert-butyl ester

To a stirred solution of4-benzyl-3-[2-(3-bromo-phenyl)-acetyl]-oxozolidin-2-one (4.02 g, 10.7mmol) in anhydrous THF (50 mL) was added LiHMDS (16.5 mL, 16.5 mmol, 1.0M solution in THF) at −78° C. The reaction mixture was stirred for 1.5 hat −78° C., and then tert-butyl-4-bromo methyl benzoate (3.75 g, 11.8mmol, in THF 10 mL) was added dropwise, stirred for 2 h at −78° C. andthen allowed to warm to rt for 1 h. After completion of the reactionquenched with saturated NH₄Cl solution (100 mL) and stirred for 10 min.The reaction mixture was extracted with ethyl acetate (2×250 mL) and theorganic layer was washed with brine, dried over Na₂SO₄ and concentratedunder reduced pressure. The crude product was precipitated from minimumamount of EtOAc and hexane at room temperature to afford4-{3-(4-Benzyl-2-oxo-oxazolidin-3-yl)-2-[4-(3-bromophenyl)-3-oxo-propyl}-benzoicacid tert-butyl ester as a yellow solid ¹H NMR (300 MHz, CDCl₃): δ 7.75(d, J=6.3 Hz, 2H), 7.44 (d, 0.1=2.1 Hz, 2H), 7.26-7.04 (m, 9H), 6.79(dd, J=1.8, 5.7 Hz, 2H), 5.29 (dd, J=2.1, 6.3 Hz, 1H), 3.89 (s, 3H),3.83 (d, J=7.5 Hz, 1H), 3.41 (dd, J=8.4, 13.8 Hz, 1H), 3.05 (dd. J=7.2,13.5 Hz, 1H); TLC conditions: Uniplate silica gel, 250 microns; mobilephase=ethyl acetate-hexanes (1:4); R=0.45.

Step 3: 4-[2-carboxy-2-(3-bromo-phenyl)-ethyl]-benzoic acid tert-butylester (5)

To a stirred solution of4-{3-(4-benzyl-2-oxo-oxazolidin-3-yl)-2-[4-(3-bromophenyl)-3-oxo-propyl}-benzoicacid tert-butyl ester (2.3 g, 3.7 mmol) in THF/H₂O (20 mL) (3:1) at roomtemperature were added H₂O₂ (1.25 g, 37.0 mmol 35% in H₂O) followed byLiOH (0.62 g, 14.8 mmol). The reaction mixture was stirred for 3 h, atroom temperature and quenched with 0.1 N HCl. The reaction mixture wasextracted with ethyl acetate (100 mL) and dried over MgSa₄ filtered andconcentrated under reduced pressure to afford the crude acid. This crudeproduct was purified by column chromatography on silica gel, elutingwith CH₂Cl₂/MeOH 2%-15% to afford4-[2-carboxy-2-(3-bromo-phenyl)-ethyl]-benzoic acid tert-butyl ester(1.5 g, 75%). ¹H NMR (300 MHz, DMSO-d₆): δ 12.52 (s, 1H), 7.77 (d, J=8.4Hz, 2H), 7.48 (d, J=8.7 Hz, 2H), 7.28 (d, J=8.4 Hz, 2H), 7.22 (d, J=8.4Hz, 2H), 3.93 (t, J=7.8 Hz, 1H), 3.30 (dd, J=8.4, 13.8 Hz, 1H), 3.0 (dd,J=8.1, 13.8 Hz, 1H), 1.49 (s, 9H), TLC conditions: Uniplate silica gel,250 microns; mobile phase=CH₂Cl₂/MeOH (10%); R_(f)=0.4. Chiral HPLCconditions: Kromasil 100-5-TBB chiral column 250×4.6 cm, (5%hexane/2-propanol to 30%), 35 min, flow rate 1 mL/min, RT=12.41 min(enantiomeric 0.10 excess: >96%)

Step 4: 4′-Chloro-2′-methyl-biphenyl-4-amine

A mixture of 4-iodo-aniline (25.0 g, 114.1 mmol),2-methyl-4-chlorophenyl-boronic acid (29.17 g, 171.1 mmol),PdCl₂(P(o-tolyl)₃)₂ (11.66 g, 14.8 mmol), and Na₂CO₃ (60.49 g, 570.7mmol) in DME/EtOH/H₂O (100/50/25 mL) was heated 125° C. for 2 h. Thereaction mixture was cooled to room temperature, filtered and washedwith EtOAc (200 mL). The solvent was removed under reduced pressure. Thecrude mixture was extracted with ethyl acetate (500 mL) and the organiclayer was washed with brine, dried over Na₂SO₄ and concentrated underreduced pressure. The resulting crude was purified by columnchromatography on silica gel, eluting with 5-30% Hexane/EtOAc to afford4′-Chloro-2′-methyl biphenyl-4-ylamine. ¹H NMR (300 MHz, CDCl₃): δ7.24-7.08 (m, 5H), 6.72 (d, J=7.2 Hz, 2H), 3.70 (bs, 2H), 2.27 (s, 3H).

Step 5:4-[2-(3-Bromo-phenyl)-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoicacid tort-butyl ester

To a stirred suspension of4-[2-carboxy-2-(3-bromo-phenyl)-ethyl]-benzoic acid tert-butyl ester(2.41 g, 5.94 mmol) in anhydrous CH₂Cl₂ (20 mL), was addedoxalylchloride (1.0 mL, 11.8 mmol) at room temperature The reactionmixture was stirred for 14 h, concentrated under reduced pressure andazeotroped with CH₂Cl₂ (2×10 mL). The crude acid chloride (2.2 g, 1.61mmol) was treated with 4-chloro-2-methyl biphenyl-4-ylamine (1.24 g,5.71 mmol) and N,N-diispropylethylamine (2.53 mL, 15.5 mmol) in CH₂Cl₂(25 mL) at 0° C. The reaction mixture was stirred for 14 h at roomtemperature and concentrated under reduced pressure. The crude productwas purified by column chromatography on silica gel, elutin withCH₂Cl₂-hexanes (30%-100%) to give4-[2-(3-bromo-phenyl)-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoicacid tert-butyl ester as a brownish solid (2.4 g, 88%). ¹H NMR (500 MHz,CDCl₃): δ 7.74 (d, J=14.0 Hz, 2H), 7.58-7.49 (m, 4H), 7.39-7.22 (m, 4H),7.24-7.19 (m, 3H), 7.14 (d, J=14.0 Hz, 2H), 4.03 (t, J=11.0 Hz, 1H),3.42 (dd, J=15.5, 22.5 Hz, 1H), 3.03 (dd, J=11.0, 22.5 Hz, 1H), 2.17 (s,3H), 1.49 (s, 9H); Chiral HPLC conditions: Chiralcel OD-H T=23° C.;mobile phase=5-25% hexane/IPA; flow rate=1.0 min; detection=254, 280,220 nm retention time in min: 16.66 min (enantiomeric excess: 97.3%).

Step 6:4-{2′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoicacid tert-butyl ester

To4-[2-(3-bromo-phenyl)-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoicacid tert-butyl ester (1.2 g, 1.98 mmol) in DME (30 mL), was added4,4-dimethyl-cyclo-hex-1-enyl-boronic acid (0.76 g, 4.96 mmol),PdCl₂(P(o-tolyl)₃)₂ (202 mg, 0.25 mmol), and diisopropylethylamine (1.0mL, 5.94 mmol). The resulting mixture was heated at 85° C. for 2 h,allowed to cool to room temperature and filtered. Partitioned thefiltrate between EtOAc (20 mL) and water. The organic phase was washedwith brine, dried over sodium sulfate and concentrated under reducedpressure. The resulting crude was purified by column chromatography onsilica gel, eluting with CH₂Cl₂:hexanes (20%-100%) to afford4-{4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoicacid tert-butyl ester as a yellow solid. ¹H NMR (500 MHz, CDCl₃): δ10.14 (s, 1H), 7.76 (d. J=8.5 Hz, 2H), 7.57 (d, J=8.4 Hz, 2H), 7.48 (s,1H), 7.35-7.20 (m, 9H), 7.13 (d, J=8.5 Hz, 1H), 6.06 (bt, 1H), 4.03 (t,J=6.5 Hz, 1H), 3.48 (dd, J=4.5, 13.5 Hz, 1H), 3.04 (dd, J=6.5, 14.0 Hz,1H), 2.37-2.34 (m, 2H), 2.23 (t, J=7.0 Hz, 1H), 2.18 (s, 3H), 1.57 (t,J=6.5 Hz, 2H), 1.50 (s, 9H), 1.47 (t, J=6.5 Hz, 1H), 0.93 (s, 6H).

Step 7:4-{2′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoicacid

To a stirred solution of4-{4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoicacid tert-butyl ester (0.82 g, 1.29 mmol) in CH₂Cl₂ (30 mL) at roomtemperature, added trifluoroacetic acid (2.5 ml), and conc HCl (1.0 mL)The reaction mixture was stirred overnight. The organic solvents wereremoved under reduced pressure. The residue was extracted with ethylacetate (2×100 mL), dried over MgSO₄ and concentrated o afford4-{2′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoicacid as a solid, ¹H NMR (500 MHz, DMSO-d₆): 10.05 (s, 1H), 7.73 (d, =8.0Hz, 2H), 7.48 (d, J=8.5 Hz, 2H), 7.40 (s, 1H), 7.29-7.05 (m, 10H), 5.99(s, 1H), 3.96 (t, J=10.5 Hz, 1H), 2.95 (dd, J=5.0, 14.0 Hz, 1H),2.20-2.18 (m, 1H), 2.14 (t, J=7.5 Hz, 1H), 2.09 (s, 3H), 1.89 (bs, 2H),1.49 (t, J=7.5 Hz, 1H), 1.39 (t, J=6.0 Hz, 2H), 0.84 (s, 6H). ChiralHPLC conditions: Chiralcel OD-H T-23° C.; mobile phase=10-30%hexane/IPA; flow rate=1.0 mL/min; detection=254, 280, 220 nm retentiontime in min: 19.81 min (enantiomeric excess: 70.8%)

Step 8:4-{2′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohexyl)-phenyl]-ethyl}-benzoicacid

A stirred solution of4-{4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoicacid (0.62 g, 1.07 mmol) in ethyl acetate (30 mL) at room temperature,was added Pd/C (100 mg). The mixture was stirred under 1 atm of H₂ (gas)at room temperature for 4 h. The catalyst was removed by filtrationthrough a Celite plug and washed with ethyl acetate (2×50 mL).Concentration of the filtrate afforded4-{2′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoicacid as a solid. ¹H NMR (300 MHz, DMSO-d₆): 12.69 (s, 1H), 10.06 (s,1H), 7.72 (d, J=7.0 Hz, 2H), 7.49 (d, J=7.0 Hz, 2H), 7.27-7.03 (m, 11H),3.94 (t, J=5.5 Hz, 1H), 3.39 (t, J=11.5 Hz, 1H), 2.93 (dd, J=5.5, 13.0Hz, 1H), 2.16 (t, J=7.0 Hz, 1H), 2.09 (s, 3H), 1.48-1.43 (m, 4H),1.24-1.19 (m, 2H), 0.87 (s, 3H), 0.84 (s, 3H).

Step 9: Sodium-2-(4-{2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-2-[3-(4,4-dimethyl-cyclohexyl)-phenyl]-ethyl}-benzoylamino-ethanesulfonic acid

To a mixture of4-{2″-chloro-2″-methyl-biphenyl-4-ylcarbamoyl)-2-[(3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoicacid (0.6 g, 1.03 mmol) and EDCI (290 mg, 1.55 mmol) in DMF (7 mL),added HOBt (230 mg, 1.55 mmol), N,N-diisopropylethylamine (0.4 g, 2.06mmol), and taurine (250 mg, 0.5 mmol). The resulting mixture was stirredfor 14 h. The reaction solvent was removed under reduced pressure. Theresidue mixture was dissolved in 0.1 N NaHCO₃ and acetonitrile, purifiedby column chromatography on a C-18 silica gel flash chromatographycolumn, eluting with an acetonitrile-water gradient.Sodium-2-(4-{2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoylamino-ethanesulfonic acid was obtained as a white solid, ¹H NMR (500 MHz, DMSO-d₆):9.98 (s, 1H), 8.25 (bs, 1H), 7.50 (d, J=3.0 Hz, 2H), 7.43 (d, J=5.5 Hz,2H), 7.19-6.98 (m, 11H), 3.85 (bs, 1H), 3.32-3.18 (m, 3H), 2.83 (d,J=14.0 Hz, 2H), 2.47 (bs, 2H), 2.23 (bs, 1H), 2.03 (s, 3H), 1.42-1.15(m, 6H), 0.81 (s, 3H), 0.78 (s, 3H); LC-MS m/z=685 [C₃₉H₄₂N₂O₅S]⁺; AnalCalcd: (MF:C₃₉₈H₄₂N₂O₅SNa+3.3 H₂O) Calcd: C: 60.94, H: 6.37, N: 3.64.Found: C, 60.82; H, 6.08; N, 3.57. Chiral HPLC conditions:Regis-Whelk-01-786615, (S,S)10/100 250×10 mm T=23° C.; mobile phase=100%ACN/(59% NH₄CO₃, +H₂O) flow rate=1.0 mL/min; detection=254 nm retentiontime in min: 12.39 min (enantiomeric excess: 95.1%)

Example 3: Ammonium2-(S)-{4-[2-[4-(4-(R)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonateStep 1: [4-(4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethylester

To a mixture of 4-bromophenyl ethyl acetate (780 mg),4-t-butyl-cyclohexen-1-yl boronic acid (897 mg), PdCl₂(P(o-tolyl)₃)₂(254 mg) in THF: ethanol: water (8 mL: 4 mL: 2 mL), added sodiumcarbonate (1.377 g). The sealed flask was heated at 140° C. for a 5 minperiod. The heterogeneous mixture was treated with an excess of 1Maqueous hydrochloric acid and filtered through a celite pad. The organicsolvents were removed under reduced pressure and the residue partitionedbetween ethyl acetate and water. The organic phase was washed with waterand saturated aqueous sodium chloride, dried over magnesium sulfate andchromatographed on silica gel using an ethyl acetate/hexanes gradient.The product was obtained as a yellow oil.

¹H NMR (500 MHz, CDCl₃); 7.32 (d, J=8 Hz, 2H), 7.20 (d, J=8 Hz, 2H),6.10 (m, 1H), 4.12 (q, J=7 Hz, 2H), 3.57 (s, 2H), 2.52-2.32 (m, 2H),2.26-2.18 (m, 1H), 2-1.9 (m, 2H), 1.38-1.2 (m, 5H), 0.91 (s, 9H).

Step 2:[4-((1R,2R,4S)-4-tert-Butyl-1,2-dihydroxy-cyclohexyl)-phenyl]-aceticacid ethyl ester and[4-((1R,2R,4R)-4-tert-Butyl-1,2-dihydroxy-cyclohexyl)-phenyl]-aceticacid ethyl ester

A mixture of AD-mix-beta (6.512 g, J. Org. Chem 57, 2768 (1992)) andmethanesulfonamide (443 mg, 4.66 mmol) in tert-butanol (23 mL) and water(28 mL) was cooled to 2-4° C. To this mixture,[4-(4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethyl ester (1.4g, 4.66 mmol) in tert-Butanol (5 mL) was added slowly while making surethat the temperature remained in the 2-4° C. range. The mixture wasstirred at the same temperature for a period of 4 days then quenched byadding sodium sulfite (1.5 g/mmol starting material) in water (20 mL).After allowing to warm to room temperature, the reaction mixture wasstirred an additional 1 h before being partitioned between ethyl acetateand water. The organic phase was washed with brine then concentratedunder reduced pressure to afford crude material which was purified bychromatography on silica gel, eluting with an ethyl acetate/hexanesgradient. Two products were obtained. In agreement with the report fromHamon et al (Tetrahedron 57, 9499 (2001)) they were assigned as follows:

First eluting product:[4-((1R,2R,4R)-4-tert-Butyl-1,2-dihydroxy-cyclohexyl)-phenyl]-aceticacid ethyl ester ¹H NMR (500 MHz, CDCl₃): 7.46 (m, 2H), 7.28 (m, 2H),4.16 (q, J=7 Hz, 2H), 3.99 (m, 1H), 3.61 (s, 2H), 2.59 (m, 1H), 1.92 (m,2H), 1.64-1.46 (m, 5H), 1.27 (t, J=7 Hz, 3H), 0.93 (s, 9H).

Second eluting product:[4-((1R,2R,4S)-4-tert-Butyl-1,2-dihydroxy-cyclohexyl)-phenyl]-aceticacid ethyl ester. ¹H NMR (500 MHz, CDCl₃): 7.51 (m, 2H), 7.30 (m, 2H),4.36 (m, 1H), 4.16 (q, J=7 Hz, 2H), 3.62 (s, 2H), 2.74 (m, 1H), 2.26 (m,1H), 2.20 (m, 1H), 2.07 (m, 1H), 1.92 (m, 1H), 1.75 (m, 1H), 1.27 (t,J=7 Hz, 3H), 1.17 (m, 2H), 0.80 (s, 9H).

Step 3:[4-((3R,6R,7R)-6-tert-Butyl-2-thioxo-tetrahydro-benzo[1,3]dioxol-3-yl)-phenyl]-aceticacid ethyl ester

A solution of[4-((1R,2R,4R)-4-tert-Butyl-1,2-dihydroxy-cyclohexyl)-phenyl]-aceticacid ethyl ester (319 mg, 0.95 mmol) and thiocarbonyl diimidazole (309mg, 1.91 mmol) in THF (15 mL) was refluxed under N₂ overnight. Thereaction mixture was partitioned between ethyl acetate and brine, driedover Na₂SO₄ and concentrated under reduced pressure. Purification of theresidue by chromatography on silica gel using an ethyl acetate/hexanesgradient afforded 297 mg of product.

¹H NMR (500 MHz, CDCl₃): 7.31 (m, 4H), 4.97 (dd, J=9 Hz, J=7 Hz, 1H),4.13 (q, J=7 Hz, 2H), 3.59 (s, 2H), 2.53-2.48 (m, 1H), 2.40-2.31 (m,1H), 1.90-1.72 (m, 2H), 1.40-1.15 (m, 6H), 0.91 (s, 9H).

Step 4: [4-((R)-4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethylester

A solution of[4-((3aR,6S,7aR)-6-tert-Butyl-2-thioxo-tetrahydro-benzo[1,3]dioxol-3a-yl)-phenyl]-aceticacid ethyl ester (297 mg, 0.80 mmol) ire triethylphosphite (3 mL) wasslowly added to a solution of triethylphosphite (10 mL) heated to refluxrate of addition was such that the reaction temperature exceeded 150° C.After refluxing overnight, the solvent was removed under vacuum and thecrude reaction mixture was loaded on top of a silica column and elutedwith an ethyl acetate/hexanes gradient to afford 145 mg of the titlecompound. ¹H NMR (500 MHz, CDCl₃): 7.32 (d, J=8 Hz, 2H), 7.20 (d, J=8Hz, 2H), 6.10 (m, 1H), 4.12 (q, J=7 Hz, 2H), 3.57 (s, 2H), 2.52-2.32 (m,2H), 2.26-2.18 (m, 1H), 2-1.9 (m, 2H), 1.38-1.2 (m, 5H), 0.91 (s, 9H).

Determination of the enantiomeric excess: A sample of the product wastreated with an excess of aqueous 1M NaOH:ethanol:water (1:2:3 ratio byvolume) and heated at 125° C. for a 5 min period. The organic solventswere removed wider reduced pressure and the residue was partitionedbetween ethyl acetate and 1M aqueous HCl. The organic phase was washedwith water and a saturated sodium chloride solution and then dried overmagnesium sulfate. The enantiomeric excess of the product was determinedto be >99% by chiral HPLC utilizing a Chiral Technologies ChiralPak AD-H250 mm×4.6 mm column, eluting at a 1.0 mL/min flow rate using a mixtureof hexanes:isopropanol:methanesulfonic acid in a 95:5:0.1 ratio. Thesample was dissolved at 1 mg/mL in ethanol prior to injection. Theretention time observed was 6.2 min.

Step 5:4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-ethoxylcarbonyl-ethyl}-benzoicacid tert-butyl ester

To [4-((R)-4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethyl ester(95 mg, 0.32 mmol) in anhydrous THF (5 mL), chilled to −78° C., wasadded 380 uL (0.38 mmol) of 1M lithium hexamethyldisilazane in THF. Theresulting solution was stirred for 1 hr before 4-Bromomethylbenzoic acidtert-butyl ester (94 mg, 0.35 mmol) was added. The reaction mixture wasallowed to warm to rt overnight then quenched with saturated NH₄Clsolution. After partitioning between ethyl acetate and brine the organicportion was dried over Na₂SO₄ and concentrated under reduced pressure.Purification of the crude by prep TLC (Analtech, 2 mm silica plates)using an hexane/ethyl acetate (10:1) gave 63 mg of product. ¹H NMR (500MHz, CDCl₃): 7.83 (d, J=8.5 Hz, 2H), 7.31 (d, J=8.5 Hz, 2H), 7.20 (d,J=8 Hz, 2H), 7.14 (d, J=8 Hz, 2H), 6.12 (m, 1H), 4.03 (q, J=7 Hz 2H),3.80 (t, J=8.5 Hz, 1H), 3.41 (dd, J=14, 8.5 Hz, 1H), 3.03 (dd, J=14, 7Hz, 1H), 2.56-2.4 (m, 2H), 2.35-2.2 (m, 1H), 2.05-1.9 (m, 2M, 156 (s,9H), 1.36-1.2 (m, 2H), 1.11 (t, J=7 Hz, 3H), 0.89 (s, 9H).

Step 6:4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-carboxy-ethyl}-benzoicacid tert-butyl ester

To4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-ethoxylcarbonyl-ethyl}-benzoicacid tert-butyl ester (63 mg, 0.13 mmol) dissolved in a solution of THF(3 mL), MeOH (1 mL) and water (1 mL) was added lithium hydroxide (27 mg,0.64 mmol). The solution was stirred at rt for 5 hrs then neutralizedwith 3M KH₂PO₄ and extracted with ethyl acetate. The organic portion waswashed with brine, dried over Na₂SO₄ and concentrated under vacuum toafford crude material which was used without further purification.

Step 7:4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoicacid tert-butyl ester

To{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-carboxy-ethyl}-benzoicacid tert-butyl ester (697 rug, 1.51 mmol) in anhydrous dichloromethane:(30 mL) was added oxalyl chloride (650 uL, 7.53 mmol) and 3 drops ofDMF. The resulting solution was stirred at rt for 1 hr before beingconcentrated under vacuum. The residue was co-evaporated with toluene(1×5 mL) then dissolved in toluene again (20 mL). To the mixture wasadded 4-chloro-2-methyl biphenyl-4-ylamine (361 mg, 1.66 mmol) and DIPEA(1.3 mL, 7.53 mmol). The resulting mixture was refluxed for 90 min,diluted with ethyl acetate and washed with saturated NaHCO₃. The organicportion was dried over Na₂SO₄ and concentrated under vacuum to affordcrude material which was crystallized from MeOH to afford a white solid(590 mg). Removing the solvent and purification of the residue bychromatography on silica gel using an ethyl acetate/hexanes gradientafforded an additional 137 mg of product. ¹H NMR (500 MHz, DMSO-d₆):10.16 (s, 1H), 7.78 (d, J=8 Hz, 2H), 7.59 (d, J=8.5 Hz, 2H), 7.4-7.32(m, 7H), 7.21 (d, J=8 Hz, 2H), 7.15 (d, J=8 Hz, 2H), 6.14 (m, 1H), 4.03(dd, J=9, 7 Hz, 1H), 3.48 (dd, J=13.5, 9.5 Hz, 1H), 3.41 (dd, J=13.5,6.5 Hz, 1H), 2.4-2.3 (n, 1H), 2.2-2.15 (m, 4H), 1.96-1.9 (m, 2H), 1.52(s, 9H), 1.32-1.2 (m, 2H), 0.89 (s, 9H).

Step 8:4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoicacid

To4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoicacid tert-butyl ester (727 mg, 1.1 mmol) was added 4N HCl/dioxane (30mL), water (5 mL) and conc. HCl (1 mL). The resulting solution wasstirred at rt overnight. The excess solvent was removed under vacuum andthe residue co-evaporated with toluene to afford the desired crudeproduct as a gummy oil. The crude material was used the next stepwithout further purification.

Step 9: Sodium,2-{4-[2-[4-(4-(R)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro)-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate

To crude4-{2-[4-(4-(R)-tert-Butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoic acid (assumed 1.1 mmol) in DMF(25 mL) was added EDC (316 mg, 1.65 mmol), HOBt (252 mg, 1.65 mmol),taurine (206 mg, 1.65 mmol) and DIPEA (550 uL, 3.29 mmol). The resultingmixture was stirred at rt overnight. The excess solvent was removedunder vacuum and to the oily residue was added excess 1N HCl. Afterdecanting off the excess 1N HCl, the residue was dissolved inacetonitrile/MeOH, made basic with saturated NaHCO₃ and purified usingreverse phase flash chromatography and eluting with anacetonitrile/water gradient. The sodium salt was obtained as a whitesolid. LCMS: 711.6 [M-H]⁻

Step 10: Ammonium,2-(S)-{4-[2-[4-(4-(R)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate

Sodium,2-{4-[2-[4-(4-(R)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate(obtained on Step 8 above), was dissolved in DMF. The product wassubjected to preparative HPLC on a Pirkle Covalent (S,S)-Whelk-01 column(250 mm×10 mm), eluting at 10 mL/min with a gradient of acetonitrile and5 mM ammonium bicarbonate. The title compound was the first of the twodiastereomers to elute. Conditions fir the determination of theenantiomeric excess by HPLC: Regis-Whelk-01-786615, (S,S)10/100 250×10mm T=23° C.; mobile phase=100% ACN/(5% Phosphate pH 7.0, ACN) flowrate=1.0 mL/min; detection=254 nm. Retention time in min: 18.22 min(enantiomeric excess: 99.1%). ¹H NMR (500 MHz, CD₃OD): 7.71 (d, J=8.5Hz, 2H), 7.51 (d, J=8.5 Hz, 2H), 7.4-725 (m, 7H), 7.20 (d, J=8.5 Hz,2H), 7.13 (d, J=8.5 Hz, 2H), 3.97 (dd, J=9.5, 6.5 Hz, 1H), 3.78 (t,J=6.5 Hz, 2H), 3.52 (dd, J=13.5, 9.5 Hz, 0.1 H), 3.10 (dd, J=13.5, 6.5Hz, 1H), 3.07 (t, J=6.5 Hz, 2H), 2.5-2.3 (m, 2H), 2.3-2.20 (m, 4H),2.1-1.95 (m, 2H), 1.38 (s, 10H), 0.94 (s, 9H).

Example 4: Ammonium,2-(R)-{4-[2-[4-(4-(R)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate

The title compound was the second compound eluting from the chiralchromatography reported in Example 1 Step 9. Conditions for thedetermination of the enantiomeric excess by HPLC: Regis-Whelk-01-786615,(S,S)10/100 250×10 mm T=23° C.; mobile phase=100% ACN/(5% PhosphatepH=7.0, ACN) flow rate=1.0 mL/min; detection=254 nm. Retention time inmin: 23.55 min (enantiomeric excess: 99.5%). ¹H NMR (500 MHz, CD₃OD):7.71 (d, J=8.5 Hz, 2H), 7.51 (d, J=8.5 Hz, 2H), 7.4-7.25 (m, 7H), 7.20(d, J=8.5 Hz, 2H), 7.13 (d, J=8.5 Hz, 2H), 3.97 (dd, J=9.5, 6.5 Hz, 1H),3.78 (t, J=6.5 Hz, 2H), 3.52 (dd, J=13.5, 9.5 Hz, 1H), 3.10 (dd, J=13.5,6.5 Hz, 1H), 3.07 (t, J=6.5 Hz, 2H), 2.5-2.3 (m, 2H), 2.3-2.20 (m, 4H),2.1-1.95 (m, 2H), 1.38 (s, 10H), 0.94 (s, 9H).

Example 5: Ammonium,2-(S)-{4-[2-[4-(4-(S)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonateStep 1: [4-((S)-4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethylester

Utilizing the second eluting diol in Example 3, Step 2, the chiralalkene shown above was obtained after utilizing the methods described inExample 3, Steps 3 and 4. ¹H NMR (500 MHz, CDCl₃): 7.32 (d, J=8 Hz, 2H),7.20 (d, J=8 Hz, 2H), 6.10 (m, 1H), 4.12 (q, J=7 Hz, 2H), 3.57 (s, 2H),2.52-2.32 (m, 2H), 2.26-2.18 (m, 1H), 2-1.9 (m, 2H), 1.38-1.2 (m, 5H),0.91 (s, 9H).

Determination of the enantiomeric excess: A sample of the product wastreated with an excess of aqueous 1M NaOH:ethanol:water (1:2:3 ratio byvolume) and heated at 125° C. for a 5 min period. The organic solventswere removed under reduced pressure and the residue was partitionedbetween ethyl acteate and 1M aqueous NCl. The organic phase was washedwith water and a saturated sodium chloride solution and then dried overmagnesium sulfate. The enantiomeric excess of the product was determinedto be >99% by chiral HPLC utilizing a Chiral Technologies ChiralPak AD-H250 mm×4.6 mm column, eluting at a 1.0 mL/min flow rate using a mixtureof hexanes:isopropanol:methane sulfonic acid in a 95:5:0.1 ratio. Thesample was dissolved at 1 mg/mL in ethanol prior to injection. Theretention time observed was 5.6 min.

Step 2: Ammonium,2-(S)-{4-[2-[4-(4-(S)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate

[4-((S)-4-tert-Butyl-cyclohex-1-enyl)-phenyl]-acetic acid ethyl ester(Step 1, above), was utilized to yield the title compound through thesequence illustrated in Example 3, Steps 5-10. The title compound elutedfirst among the two possible diastereomers after preparative HPLC on aPirkle Covalent (S, S)-Whelk-01 column (250 mm×10 mm), eluting at 10mL/min with a gradient of acetonitrile and 5 mM ammonium bicarbonate.

Conditions for the determination of the enantiomeric excess by HPLC: Thesample was diluted in ethanol at a 1 mg/mL concentration. It wasinjected into an HPLC system equipped with a Regis-Whelk-01-786615,(S,S)10/100 250×10 mm column kept at 23° C. The column was eluted with agradient of two solvents A and B for a 30 min period. The composition ofthe gradient ranged from 40% A to 65% A over this period (with thebalance of the solvent being B). Solvent A was acetonitrile, solvent Bwas a mixture of 5% acetonitrile and 95% water containing 5 mM potassiumphosphate monobasic (pH 6.8). The product was detected by UV at 254 nm.Retention time in min: 18.23 min (enantiomeric excess: >99.5%). ¹H NMR(500 MHz, CD₃OD): 7.71 (d, J=8.5 Hz, 2H), 7.5.1 (d, J=8.5 Hz, 2H),7.4-7.25 (m, 7H), 7.20 (d, J=8.5 Hz, 2H), 7.13 (d, J=8.5 Hz, 2H), 3.97(dd, J=9.5, 6.5 Hz, 1H), 178 (t, J=6.5 Hz, 2H), 3.52 (dd, J=13.5, 9.5Hz, 1H), 3.10 (dd, J=13.5, 6.5 Hz, 1H), 3.07 (t, 6.5 Hz, 2H), 2.5-2.3(m, 2H), 2.3-2.20 (m, 4H), 2.1-1.95 (m, 2H), 1.38 (s, 10H), 0.94 (s,9H).

Example 6: Ammonium,2-(R)-{4-[2-[4-(4-(S)-tert-butylcyclohex-1-enyl)-phenyl]-2-(4′-chloro-2′-methylbiphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonate

The title compound was the second compound eluting from the chiralchromatography reported in Example 5, Step 2. The sample was diluted inethanol at a 1 mg/mL concentration. It was injected into an HPLC systemequipped with a Regis-Whelk-01-786615, (S,S)10/100 250×10 mm column keptat 23° C. The column was eluted with a gradient of two solvents A and Bfor a 30 min period. The composition of the gradient ranged from 40% Ato 65% A over this period (with the balance of the solvent being B).Solvent A was acetonitrile, solvent B was a mixture of 5% acetonitrileand 95% water containing 5 mM potassium phosphate monobasic (pH 6.8).The product was detected by UV at 254 nm. Retention time in min: 23.41min (enantiomeric excess: 99.5%). ¹H NMR (500 MHz. CD₃OD): 7.71 (d,J=8.5 Hz, 2H), 7.51 (d, J=8.5 Hz, 2H), 7.4-7.25 (m, 7H), 7.20 (d, J=8.5Hz, 2H), 7.13 (d, J=8.5 Hz, 2H), 3.97 (dd, J=9.5, 6.5 Hz, 1H), 3.78 (t,J=6.5 Hz, 2H), 3.52 (dd, J=13.5, 9.5 Hz, 1H), 3.10 (dd, J=13.5, 6.5 Hz,1H), 3.07 (t, J=6.5 Hz, 2H), 2.5-2.3 (m, 2H), 2.3-2.20 (m, 4H), 2.1-1.95(m, 2H), 1.38 (s, 10H), 0.94 (s, 9H).

Using the methods described in Examples 1-6, the following compoundswere synthesized.

Example 7:Sodium-2-(R)-4-[2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(4-(3,3-dimethyl-but-1-enyl)-phenyl]-ethyl}-benzoylamino ethane sulfonic acid

¹H NMR (500 MHz, DMSO-d₆): 10.14 (s, 1H), 8.39 (t, J=9.8 Hz, 1H), 7.61(d, J=8.7 Hz, 2H), 7.55 (d, J=8.7 Hz, 2H), 7.33-7.12 (m, 11H), 6.25 (dd,J=16.2, 3.6 Hz, 2H), 3.98 (t, J=8.4 Hz, 1H), 3.49-3.38 (m, 3H), 3.0 (dd,J=6.3, 7.8 Hz, 1H), 2.61 (t, J=7.8 Hz, 2H), 2.17 (s, 3H), 1.06 (s, 3H);LC-MS m/z=657 [C₃₇H₃₈N₂O₅SClNa+H]⁺; HPLC conditions: Waters AtlantisC-18 OBD 4.6×150 mm; mobile phase=ACN/(H₂O: 0.1 TFA) flow rate=1.0mL/min; detection=UV@254, 220 nm retention time in min: 12.33; AnalCalcd: (MF: C₃₇H₃₈N₂O₅SClNa+1.2H₂O) Calcd: C: 63.23, H: 5.79, N: 3.99.Found: C, 63.02; H, 5.89; N, 4.15.

Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mLconcentration. It was injected into an HPLC system equipped with aRegis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. Thecolumn was eluted with a gradient of two solvents A and B for a 30 minperiod. The composition of the gradient ranged from 40% A to 65% A overthis period (with the balance of the solvent being B). Solvent A wasacetonitrile, solvent B was a mixture of 5% acetonitrile and 95% watercontaining 5 mM potassium phosphate monobasic (pH 6.8). The product wasdetected by UV at 254 nm. Retention time in min: 14.08 min (enantiomericexcess: >97.06%)

Example 8:Sodium-2-(S)-4-[2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[(4-(3,3-dimethyl-but-1-enyl)-phenyl]-ethyl}-benzoylamino ethane sulfonic acid

¹H NMR (500 MHz, DMSO-d₆): 10.14 (s, 1H), 8.39 (t, J=9.8 Hz, 1H), 7.61(d, J=8.7 Hz, 2H), 7.56 (d, J=6.6 Hz, 2H), 7.33-7.12 (m, 1H), 6.26 (dd,J=16.2, 3.6 Hz, 2H), 3.98 (t, J=9.9 Hz, 1H), 3.49-3.39 (m, 3H), 3.0 (dd,J=6.3, 7.8 Hz, 1H), 2.61 (t, J=7.2 Hz, 2H), 2.17 (s, 3H), 1.05 (s, 3H);LC-MS m/z=657 [C₃₇H₃₈N₂O₅SClNa+H]⁺; HPLC conditions: Waters AtlantisC-18 OBD 4.6×150 mm; mobile phase=ACN/(H₂O:0.1 TFA) flow rate=1.0mL/min; detection=UV@254, 220 nm retention time in min: 12.33; AnalCalcd: (MF: C₃₇H₃₈N₂O₅SClNa+1.5H₂O) Calcd: C: 62.75, H: 5.83, N: 3.96.Found: C, 62.65; H, 5.81; N, 4.13.

Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mLconcentration. It was injected into an HPLC system equipped with aRegis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. Thecolumn was eluted with a gradient of two solvents A and B for a 30 minperiod. The composition of the gradient ranged from 40% A to 65% A overthis period (with the balance of the solvent being B). Solvent A wasacetonitrile, solvent B was a mixture of 5% acetonitrile and 95% watercontaining 5 mM potassium phosphate monobasic (pH 6.8). The product wasdetected by UV at 254 nm. Retention time in min: 17.69 min (enantiomericexcess: >97.4%).

Example 9:2-(4-{(R)-2-(4′-Chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[4-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoylamino)-ethanesulfonicacid

¹H NMR (500 MHz, DMSO-d₆): δ 10.14 (s, 1H), 8.40 (t, J=2.8 Hz, 1H), 7.64(d, J=8.4 Hz, 2H), 7.57 (d, J=8.9 Hz, 2H), 7.37-7.14 (m, 11H), 6.07 (bs,1H), 4.0 (t, J=5.5 Hz, 1H), 3.47-3.45 (m, 3H), 3.0-2.85 (m, 1H), 2.62(t, J=4.2 Hz, 2H), 2.35 (t, j=1.2 Hz, 2H), 2.18 (s, 3H), 1.95 (t, J=2.8Hz, 2H), 1.46 (t, J=2.8 Hz, 2H), 0.91 (s, 6H), LC-MS m/z=685[C₃₉H₄₀N₂O₄SCl+H]⁺.

Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mLconcentration. It was injected into an HPLC system equipped with aRegis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. Thecolumn was eluted with a gradient of two solvents A and B for a 30 minperiod. The composition of the gradient ranged from 40% A to 70% A overthis period (with the balance of the solvent being B). Solvent A wasacetonitrile, solvent B was a mixture of 5% acetonitrile and 95% watercontaining 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO₂). Theproduct was detected by UV at 254 nm. Retention time in min: 17.92 min(enantiomeric excess: 99.5%).

Example 10:2-(4-{(S)-2-(4′-Chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-2-[4-(4,4-dimethyl-cyclohex-1-enyl)-phenyl]-ethyl}-benzoylamino)-ethanesulfonicacid

¹H NMR (500 MHz, DMSO-d₆): δ 10.15 (s, 1H), 8.40 (t, J=2.8 Hz, 1H), 7.64(d, J=8.4 Hz, 2H), 7.57 (d, J=8.9 Hz, 2H), 7.37-7.11 (m, 11H), 6.07 (bs,1H), 4.0 (t, J=5.5 Hz, 1H), 3.47-3.45 (m, 3H), 3.0-2.85 (m, 1H), 2.62(t, J=8.4 Hz, 2H), 2.35 (t, J=0.2 Hz, 2H), 2.18 (s, 3H), 1.95 (t, J=2.8Hz, 2H), 1.46 (t, J=2.8 Hz, 2H), 0.91 (s, 6H). LC-MS m/z=684[C₃₉H₄₀N₂O₄SCl]⁺.

Chiral HPLC conditions; The sample was diluted in ethanol at a 1 mg/mLconcentration. It was injected into an HPLC system equipped with aRegis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. Thecolumn was eluted with a gradient of two solvents A and B for a 30 minperiod. The composition of the gradient ranged from 40% A to 70% A overthis period (with the balance of the solvent being B). Solvent A wasacetonitrile, solvent B was a mixture of 5% acetonitrile and 95% watercontaining 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO₂). Theproduct was detected by UV at 254 nm. Retention time in min: 13.87 min(enantiomeric excess: 97.6%).

Example 11:Sodium-2-[4-(S)-2-(4′-tert-butyl-biphenyl-4-yl)-2-(4′-chloro-2′-methyl-phenyl-carbamoyl)-ethyl}-benzoylamino-ethanesulfonic acid

¹H NMR (500 MHz, DMSO-d₆): δ 7.70 (d, J=6.0 Hz, 2H), 7.57-7.47 (m, 11H),7.33 (d, J=8.4 Hz, 2H), 7.21-7.13 (m, 4H), 4.0 (t, J=6.0 Hz, 1H), 3.74(t, J=13.5 Hz, 2H), 3.53 (dd, J=6.3, 3.0 Hz, 1H), 3.15 (dd, J=6.6, 2.8Hz, 1H), 3.06 (t, J=6.9 Hz, 2H), 2.20 (s, 3H), 1.34 (s, 9H), LC-MSm/z=709 [C₃₅H₃₆N₂O₅SClNa]⁺.

Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mLconcentration. It was injected into an HPLC system equipped with aRegis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. Thecolumn was eluted with a gradient of two solvents A and B for a 30 minperiod. The composition of the gradient ranged from 40% A to 70% A overthis period (with the balance of the solvent being B). Solvent A wasacetonitrile, solvent B was a mixture of 5% acetonitrile and 95% watercontaining 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO₂). Theproduct was detected by UV at 254 nm. Retention time in min: 23.87 min(enantiomeric excess: 99.5%).

Example 12:Sodium-2-[4-(R)-2-(4′-tert-butyl-biphenyl-4-yl)-2-(4′-chloro-2′-methyl-phenyl-carbamoyl)-ethyl}-benzoylamino-ethanesulfonic acid

¹H NMR (500 MHz, DMSO-d₆): δ 7.70 (d, J=6.0 Hz, 2H), 7.57-7.47 (m, 11H),7.33 (d, J=8.4 Hz, 2H), 7.21-7.13 (m, 4H), 4.0 (t, J=6.0 Hz, 1H), 3.74(t, J=13.5 Hz, 2H), 3.53 (dd, J=6.3, 3.0 Hz, 1H), 3.15 (dd, J=6.6, 2.8Hz, 1H), 3.06 (t, J=6.9 Hz, 2H), 2.20 (s, 3H), 1.34 (s, 9H). LC-MSm/z=707 [C₃₅H₃₆N₂O₅SClNa-2]⁺.

Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mLconcentration. It was injected into an HPLC system equipped with aRegis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. Thecolumn was eluted with a gradient of two solvents A and B for a 30 minperiod. The composition of the gradient ranged from 40% A to 70% A overthis period (with the balance of the solvent being B). Solvent A wasacetonitrile, solvent B was a mixture of 5% acetonitrile and 95% watercontaining 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO₂). Theproduct was detected by UV at 254 nm. Retention time in min: 23.86 min(enantiomeric excess: >96.9%).

Example 13:Sodium-2-[4-(R)-2-(4′-tert-butyl-biphenyl-4-yl)-2-(2′,4′,6′-trimethyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino-ethanesulfonic acid

¹H NMR (500 MHz, DMSO-d₆): δ 7.65 (d, J=7.5 Hz, 2H), 7.51 (d, J=8.0 Hz,2H), 7.48-7.43 (m, 11H), 7.38 (d, J=8.4 Hz, 2H), 7.28 (d, J=8.4 Hz, 2H),6.92 (d, J=8.5 Hz, 2H), 6.80 (s, 1H), 3.97 (t, J=6.5 Hz, 1H), 3.71 (t,7.0 Hz, 2H), 3.50 (dd, J=9.0, 13.5 Hz, 1H), 3.08 (dd, J=6.6, 13.5 Hz,1H), 2.99 (t, J=6.5 Hz, 2H), 2.19 (s, 3H), 1.86 (s, 6H), 1.28 (s, 9H),LC-MS m/z=702 [C₄₃H₄₅N₂O₅SCl]⁺; Anal Calcd: (MF:C₄₃H₄₅NO₅SClNa+1.2H₂O+0.1 NaHCO₃) Calcd: C: 68.57, H: 6.34, N: 3.71.Found: C, 68.24; H, 5.98; N, 3.58.

Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mLconcentration. It was injected into an HPLC system equipped with aRegis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. Thecolumn was eluted with a gradient of two solvents A and B for a 30 minperiod. The composition of the gradient ranged from 40% A to 70% A overthis period (with the balance of the solvent being B). Solvent A wasacetonitrile, solvent B was a mixture of 5% acetonitrile and 95% watercontaining 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO₂). Theproduct was detected by UV at 254 nm. Retention time in min: 28.908 min(enantiomeric excess: >98.79%).

Example 14:Ammonium-2-(R)-(4-{2-[4-tert-butyl-biphenyl-4-yl)-3-[3-(4,4-dimethyl-cyclohex-1-enyl)-phenyl-4-ylcarbamoyl]ethyl}-benzoylamino-ethanesulfonic acid

¹H NMR (500 MHz, DMSO-d₆): δ 10.07 (s, 1H), 8.39 (t, J=5.0 Hz, 1H), 7.64(d, J=8.0 Hz, 2H), 7.57 (d, J=8.0 Hz, 2H), 7.54 (d, J=7.5 Hz, 2H),7.50-7.44 (m, 6H), 7.32-7.29 (m, 4H), 6.0 (bs, 1H), 4.05 (dd, J=6.0, 8.5Hz, 1H), 3.48 (dd, J=7.0, 12.5 Hz, 3H), 3.05 (dd, J=6.0, 13.5 Hz, 1H),2.63 (t, J=7.0 Hz, 2H), 2.18 (bs, 3H), 1.94 (bs, 2H), 1.44 (t, J=12.0Hz, 2H), 1.29 (s, 9H), 0.90 (s, 6H), LC-MS m/z=692 [C₄₂H₄₈N₂O₅S]⁺; AnalCalcd: (MF: C₄₂H₄₈N₂O₅S+2.8H₂O+0.6 NH₃) Calcd: C: 66.94, H: 7.41, N:4.83. Found: C, 66.90; H, 7.20; N, 4.44.

Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mLconcentration. It was injected into an HPLC system equipped with aRegis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. Thecolumn was eluted with a gradient of two solvents A and B for a 30 minperiod. The composition of the gradient ranged from 40% A to 70% A overthis period (with the balance of the solvent being B). Solvent A wasacetonitrile, solvent B was a mixture of 5% acetonitrile and 95% watercontaining 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO₂). Theproduct was detected by UV at 254 nm. Retention time in min: 13.87 min(enantiomeric excess: >99.0%).

Example 15:2-(4-[2-(4-Benzooxazol-2-yl-phenylcarbamoyl)-2-4-(1R,4R)-1,7,7-trimethyl-bicyclo[2.2.1]hept-2-en-2-yl)-phenyl]-ethyl-benzoylamino)-ethanesulfonic acid

¹H NMR (500 MHz, DMSO-d₆): δ 8.13 (d, J=8.5 Hz, 2H), 7.74-7.65 (m, 4H),7.40-7.38 (m, 6H), 7.33 (d, J=8.0 Hz, 2H), 7.21 (d, J=8.0 Hz, 2H), 5.97(d, J=3.5 Hz, 2H), 4.05 (t, J=6.0, Hz, 1H), 3.77 (t, J=6.5, Hz, 1H),3.56-3.46 (m, 1H), 3.14 (dd, J=6.0, 14.0 Hz, 1H), 3.06 (t, J=6.5 Hz,2H), 2.37 (t, J=3.5 Hz, 2H), 1.99-1.93 (m, 2H), 1.72-1.67 (m, 2H),1.33-1.29 (m, 2H), 1.15-1.09 (m, 4H), 0.90 (s, 3H), 0.88 (s, 3H). LC-MSm/z=703 [C₄₁H₄₁N₃O₆S]⁺. HPLC conditions: 250×10 mm T=23° C.: mobilephase=100% ACN/(H₂O/CAN+0.1 TFA) flow rate=1.0 mL/min; detection=254,280, 220 nm retention time in min: 7.39 min (95.0%).

Example 16:Sodium-2-[4-(R)-2-(4′-tert-butyl-biphenyl-4-yl)-2-(4′-chloro-3′-methyl-phenyl-carbamoyl)-ethyl}benzoylamino-ethanesulfonic acid

¹H NMR (500 MHz, DMSO-d₆): δ 7.71 (d, J=8.0 Hz, 2H), 7.59-7.44 (m, 13H),7.36-7.33 (m, 4H), 4.0 (dd, J=6.0, 9.0 Hz, 1H), 3.77 (t, J=7.0 Hz, 2H),3.56 (dd, J=9.0, 13.0 Hz, 1H), 3.14 (dd, J=6.0, 13.0 Hz, 1H), 3.06 (t,J=6.5 Hz, 2H), 2.41 (s, 3H), 1.34 (s, 9H). LC-MS m/z=731[C₃₅H₃₆N₂O₅SClNa]⁺.

Example 17:Ammonium-2-(R)-(4-{2-[4′-tert-butyl-biphenyl-4-yl)-2-(4-methyl-benzooxazol-2-yl)phenylcarbamoyl]-ethyl}-benzoylamino)-ethanesulfonic acid Step 1: 4-(4-Methyl-benzooxazol-2-yl)-phenylamine

To a suspension of 4-amino-benzoic acid (2.0 g, 14.5 mmol) in PPA (˜85g) was added 2-amino-m-cresol (1.8 g, 15.3 mmol). The reaction washeated to 160° C. for 14 h, then carefully quenched in aqueous sodiumcarbonate (˜50% saturated) at room temperature. Ethyl acetate was added,and the organic layer was washed with water and brine, and dried oversodium sulfate. The crude product was obtained was subsequently purifiedby flash column chromatography on silica gel eluting with ethyl acetatein hexanes to afford the desired product,4-(4-methyl-benzooxazol-2-yl)-phenylamine as a light pink solid, 1.8 g(56%). LC-MS m/z=225 [C₁₄H₁₂N₂O+H]⁺.

Step 2

The methods described in Examples 1-6 were used to generate the titlecompound from 4-(4-Methyl-benzooxazol-2-yl)-phenylamine

¹H NMR (500 MHz, DMSO-d₆): δ 10.48 (s, 1H), 8.41 (t, J=3.0 Hz, 1H), 8.12(d, J=5, 4 Hz, 2H), 7.77 (d, J=5.1 Hz, 2H), 7.67-7.46 (m, 11H), 7.34 (d,J=5.1 Hz, 1H), 7.25 (dd, J=4.5 Hz, 1H), 7.17 (d, J=4.8 Hz, 1H), 4.13 (t,J=6.0 Hz, 1H), 3.53-3.41 (m, 3H), 3.11 (dd, J=3.0, 6.5 Hz, 1H), 2.62 (t,J=3.9 Hz, 2H), 2.55 (s, 3H), 1.29 (s, 9H). LC-MS m/z=717 [C₄₂H₄₁N₃O₆S]⁺.

Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mLconcentration. It was injected into an HPLC system equipped with aRegis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. Thecolumn was eluted with a gradient of two solvents A and B for a 30 minperiod. The composition of the gradient ranged from 40% A to 70% A overthis period (with the balance of the solvent being B). Solvent A wasacetonitrile, solvent B was a mixture of 5% acetonitrile and 95% watercontaining 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO₂). Theproduct was detected by UV at 254 nm. Retention time in min: 26.69 min(enantiomeric excess: >99.5%).

Example 18:Ammonium-2-(S)-(4-{2-[4′-tert-butyl-biphenyl-4-yl)-2-(4-methyl-benzooxazol-2-yl)phenylcarbamoyl]ethyl}-benzoylamino)-ethanesulfonate

This compound was generated using the methods described in Example 19.¹H NMR (500 MHz, DMSO-d₆): δ 10.48 (s, 1H), 8.41 (t, J=3.3 Hz, 1H), 8.11(d, J=5.1 Hz, 2H), 7.78 (d, J=2.1 Hz, 2H), 7.67-7.46 (m, 11H), 7.34 (d,J=5.1 Hz, 1H), 7.25 (dd, J=4.8 Hz, 1H), 7.19 (d, J=4.8 Hz, 1H), 4.13 (t,J=6.0 Hz, 1H), 3.51-3.37 (m, 3H), 3.09 (dd, J=3.9, 4.5 Hz, 1H), 2.62 (t,J=1.2 Hz, 2H), 2.60 (s, 3H), 1.29 (s, 9H). LC-MS m/z=717 [C₄₂H₄₁N₃O₆S]⁺.

Chiral HPLC conditions: The sample was diluted in ethanol at a 1 mg/mLconcentration. It was injected into an HPLC system equipped with aRegis-Whelk-01-786615, (S,S)10/100 250×10 mm column kept at 23° C. Thecolumn was eluted with a gradient of two solvents A and B for a 30 minperiod. The composition of the gradient ranged from 40% A to 70% A overthis period (with the balance of the solvent being B). Solvent A wasacetonitrile, solvent B was a mixture of 5% acetonitrile and 95% watercontaining 5 mM ammonium bicarbonate (pH adjusted to 6.5 with CO₂). Theproduct was detected by UV at 254 nm. Retention time in min: 26.48 min(enantiomeric excess: >99.8%).

Example 19:2-{4-[(R)-2-[4-(4-(cis)-tert-Butylcyclohexyl)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonicacid

Step 1: 4-Chloromethyl benzoic acid tert-butyl ester

Oxalyl chloride (101 mL) was added dropwise over a 30 min period to aslurry of 4-chloromethyl benzoic acid (181.8 g) in dichloromethane (1.2L) containing 5 mL of DMF. After the addition was complete the reactionmixture was stirred at room temperature for 24 h, concentrated underreduced pressure and then co-evaporated with toluene. To the residue wasadded 908 mL of MTBE and the mixture was cooled to −5° C. A solution ofpotassium tert-butoxide in THF (1.0 M, 1172 mL) was added dropwiseensuring that the internal temperature remained below 10° C. After theaddition was complete, the reaction mixture was stirred for anadditional 1 hour then treated with 500 mL of saturated sodiumbicarbonate solution. After stirring 5 minutes, then settling, theorganic phase was separated, washed with saturated sodium chloridesolution and dried over magnesium sulfate. Concentration yielded 241.7 g(86% yield) as a dark oil. HNMR: CDCl₃, 1.59 ppm (s, 9H), 4.61 (s, 2H),7.45 (d, 2H), 7.99 (d, 2H)

Step 2: 4-Iodomethyl benzoic acid tert-butyl ester

Sodium iodide (229.2 g) was added to a solution of 4-chloromethylbenzoic acid tell-butyl ester (315.2 g) in acetone (1.5 L). The reactionmixture was heated to reflux for about 2 h and then allowed to cool toroom temperature. The precipitate was removed by filtration and thefiltrate concentrated under reduced pressure. The residue waspartitioned between water (500 mL) and MTBE (1500 The organic phase waswashed with saturated sodium bicarbonate and dried over magnesiumsulfate. Concentration under reduced pressure afforded 442.2 g (97%yield) dark oil. HNMR: CDCl₃, 1.59 ppm (s, 4.47 (s, 2H), 7.42 (d, 2H),7.91 (d, 2H)

Step 3: 4-Bromophenyl acetic acid methyl ester

Sulfuric acid (56.5 mL) was very slowly added to a solution of 206.6 gof 4-bromophenyl acetic acid in methanol (800 mL). After completion ofthe addition, the mixture was heated to reflux for 2 h. The refluxcondenser was replaced by a distillation head and 400 mL of methanol wasatmospherically distilled. The temperature was the reduced to 50° C. andthe reaction stirred for additional 16 h, when the mixture was thencooled to room temperature and partitioned between dichloromethane (1 L)and water (600 mL). The organic phase was washed with saturated sodiumbicarbonate and dried over magnesium sulfate. Concentration underreduced pressure provided 220.1 g (98% yield) colorless oil. HNMR:CDCl₃, 3.59 (s, 2H), 3.70 (s, 3H), 7.16 (d, 2H), 7.45 (d, 2H)

Step 4: 4-[2-(4-Bromo-phenyl)-2-methoxycarbonyl-ethyl]-benzoic acidtert-butyl ester

A solution of 246.63 g of methyl-4-bromophenyl acetate and 342.54 g of4-lodomethyl benzoic acid tert-butyl ester in THF (1233 mL) was cooledto −8° C. A solution of lithium hexamethyl disilazide in THF (1185 mL,1.0 M) was added dropwise ensuring that the temperature remain below −2°C. After the addition was complete, the reaction was allowed to proceedfor ˜45 min at the same temperature and then poured over a stirringmixture of ethyl acetate (2.46 L) and water (1.23 L). The organic phasewas washed with saturated ammonium chloride and then with water. Driedover magnesium sulfate and concentrated under reduced pressure to obtain450.5 g (100% yield) thick oil. HNMR: CDCl₃, 1.41 (s, 9H), 2.88-2.90 (m,1H), 3.24-3.28 (m, 1H), 3.45 (s, 3H), 3.63 (t, 1H), 6.96-6.99 (m, 4H),7.25 (d, 2H), 7.68 (d, 2H)

Step 5:(R)-2-(4-Bromo-phenyl)-3-(4-tert-butoxycarbonyl-phenyl)-propionate(S)-2-hydroxymethyl pyrrolidinium

4-[2-(4-Bromo-phenyl)-2-methoxycarbonyl-ethyl]-benzoic acid tert-butylester (769 g) was dissolved in THF (5.38 L) and water (3.85 L) andtreated with lithium hydroxide monohydrate (153.9 g). The reactionmixture was heated to 45° C. for approximately 1 hour. After allowingthe reaction to cool to 32° C. the reaction was poured into a stirringmixture of 11.6 L of ethyl acetate and 3.9 L of 1 M aqueous hydrochloricacid. The separated organic layer was washed with water, dried overmagnesium sulfate and concentrated under reduced pressure. Added ethylacetate (2.045 L) to the residue and warmed to 78° C. for 5 min fordissolution. The mixture was allowed to cool to 68° C. and treated with(S)-(+)-prolinol (90.5 mL). The solid that precipitated after cooling toroom temperature was filtered and rinsed with a cold mixture (7° C.) of1:1 ethyl acetate: heptane (740 mL). The solid isolated (232.4 g, 35%yield) was shown to have 94% enantiomeric excess (R isomer) by chiralHPLC analysis. HNMR: CDCl₃, 1.57 (s, 9H), 1.67-1.74, (m, 2H), 2.64-2.69(m, 1H), 2.76-2.81 (m, 1H), 2.94-2.99 (m, 1H), 3.14-3.19 (m, 1H),3.32-3.39 (m, 2H), 3.60-3.67 (m, 2H), 7.14-7.19 (m, 4H), 7.35 (d, 2H),7.80 (d, 2H). Conditions for chiral HPLC analysis Kromasil 100-5-TBBcolumn, 250×4.6 mm, 1 mL/min, 15% (1% AcOH/MTBE)/85% hexanes,230/240/250 nm.

To 228 g of the product above were added 684 mL of ethyl acetate. Themixture was warmed to reflux (additional 228 mL of ethyl acetate wereadded when the temperature reached 69° C. for mobility) where it washeld for about 10 min. The suspension was then allowed to cool to roomtemperature and filtered. Vacuum dried at 50° C. Product is a whitesolid (224.6 g, 98% yield) “R” enantiomer with enantiomeric excess of96.9% by HPLC analysis as described above.

Step 6: 4-[(R)-2-(4-Bromo-phenyl)-2-carboxy-ethyl-]-benzoic acidtert-butyl ester

A stirring slurry of 216.8 g of(R)-2-(4-Bromo-phenyl)-3-(4-tert-butoxycarbonyl-phenyl)-propionate(S)-2-hydroxymethyl pyrrolidinium in 2168 mL of ethyl acetate at 21° C.was treated 1084 mL of 10% aqueous formic acid. After 20 minutes, theseparated organic phase was washed with water and dried over magnesiumsulfate. The ethyl acetate solution was atmospherically displaced intoheptanes to yield the product as a granular solid. 166.3 g (96% yield)of white solid, enantiomeric excess (“R” enantiomer) of 96.9% by chiralHPLC analysis as described above. HNMR: CDCl₃, 1.50 (s, 9H), 2.96-3.00(m, 1H), 3.32-3.37 (m, 1H), 3.73 (t, 1H), 7.03-7.08 (m, 4H), 7.35 (d,2H), 7.76 (d, 2H)

Step 7:4-{(R)-2-[4-(4-tert-Butyl-cyclohex-1-enyl)-phenyl]-2-carboxy-ethyl}-benzoicacid tert-butyl ester

A mixture of 3.1 g of4-[(R)-2-(4-Bromo-phenyl)-2-carboxy-ethyl-]-benzoic acid tert-butylester (3. Step 2, above), 1.5 g of 4-t-butyl-cyclohex-1-enyl boronicacid, 644 mg of PdCl₂(P(o-tolyl)₃)₂, and 2.21 g of sodium carbonate in12 mL of DME and 6 mL of ethanol and 3 mL of water was heated to refluxfor a 16 h period. The reaction mixture was quenched with an excess ofaqueous ammonium chloride, added ethyl acetate and the heterogeneousmixture was filtered through a celite pad. The organic phase was washed(water, saturated sodium chloride), dried over magnesium sulfate andconcentrated. The residue was chromatographed on silica gel using amethanol-dichloromethane gradient to yield the carboxylic acid. HNMR(300 MHz, CDCl3, partial): 6.14 (1H, m), 1.58 (9H, s), 0.92 (9H, s).LCMS m/z=407.9 [(C₃₀H₃₈O₄+H)—C₄H₉]⁺.

Step 8:4-{(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-carboxy-ethyl}-benzoicacid tert-butyl ester

To a solution of4-{(R)-2-[4-(4-tert-Butyl-cyclohex-1-enyl)-phenyl]-2-carboxy-ethyl}-benzoicacid tert-butyl ester (3.0 g) in ethyl acetate (1.00 mL) added 10%palladium on carbon (300 mg). The mixture was stirred under a balloonfilled with hydrogen, until proton NMR indicated the disappearance ofthe olefinic signal. The reaction was filtered through a plug of celite,and the filtrate was concentrated under reduced pressure to give amixture of cis/trans isomers (in a ratio of 1:1, based on ¹H NMR). Theand trans isomers were separated by reverse phase chromatography withthe latter being cis (1.34 g, 2.9 mmol, 35%). ¹H NMR (CDCl₃): δ 0.95(9H, s), 1.23-1.38 (4H, m), 1.58 (9H, s), 1.88-1.98. (4H, m), 2.39-2.56(1H, m), 3.05-3.12 (1H, m), 3.19-3.50 (1H, m), 3.82-3.90 (1H, m),7.19-7.22 (6H, m), 8.81 (2H, d).

Based on the following reference and references within the article, thecis was assigned as described above. Garbisch, E. W.; Patterson, D. B.,J. Am. Chem. Soc., 1963, 85, 3228.

Step 9:4-[(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-(4′-chlor-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoicacid tert-butyl ester

To4-{(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-carboxy-ethyl}-benzoicacid tert-butyl ester (300 mg) in dichloromethane (10 mL) was added asolution of oxalyl chloride in dichloromethane (2.0M, 0.54 mL) followedby 2 drops of DMF. The reaction mixture was stirred at room temperaturefor 2 h and concentrated under reduced pressure. The residue wasdissolved in dichloromethane (20 mL), and treated with4′-Chloro-2′-methyl-biphenyl-4-amine (142 mg) and diisopropyl ethylamine (0.120 mL). After stirring for 1 h at room temperature, thesolvent was removed under reduced pressure and the residue treated withmethanol. The white precipitated formed was washed with methanol, driedunder vacuum and used without further purification in the followingstep.

Step 10:4-[(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-(4′-chlor-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoicacid

A solution of 431 mg of4-[(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-(4′-chlor-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoicacid tert-butyl ester in dichloromethane (10 mL) was treated withtrifluoroacetic acid (2 mL) and concentrated aqueous hydrochloric acid(1 mL). The resulting mixture was stirred for 16 h at room temperature.The organic phase was separated, washed with water and dried overmagnesium sulfate. Concentration left a residue that was used withoutfurther purification.

Step 11:2-{4-[(R)-2-[4-(4-(cis)-tert-Butyl-cyclohexy)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonicacid

(R)-4-[2-[4-(4-(cis)-tert-Butyl-cyclohexyl)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]benzoicacid (350 mg, 0.6 mmol), was taken up in 3 mL of DMF, followed byaddition of HOBt (133 mg, 0.9 mmol), EDCI (132 mg, 0.7 mmol), taurine(86 mg, 0.7 mmol) and Hunig's base (374 mg, 2.9 mmol). The resultingreaction mixture was then stirred for 16 h at room temperature. Thereaction solution was diluted with EtOAc (25 mL) and 10 mL of water,acidified with 2.4 N HCl. The layers were separated and the aqueouslayer was extracted with EtOAc (2×20 mL). The organic extracts werecombined, dried with Na₂SO₄, filtered through a frit and concentratedunder reduced pressure to give a foam. The material was subjected toreverse phase HPLC purification to give the desired product as a whitesolid (150 mg, 36%). ¹H NMR (CD₃OD): δ 0.89 (9H, s), 1.10-1.60 (6H, m),1.80-1.90 (4H, m), 2.38 (3H, s), 2.32-2.53 (1H, m), 3.05-3.10 (4H, m),3.47-3.55 (1 h, dd), 3.77 (2H, t), 3.93-3.98 (1H, m), 7.09-7.47 (11H,m), 7.50 (2H, d), 7.70 (2H, d). Anal. Calcd. For C₄₂H₄₇ClN₂O₅S+NH₃+1.2H₂O; C=65.22; H=7.13; N=5.57. Found C=65.31; H=7.00; N=5.57.

Step 12.(R)-2-{4-[2-[4-(4-(trans)-tert-Butyl-cyclohexyl)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonicacid

The trans isomer isolated from step 8,(R)-4-{2-[4-(4-(trans)-tert-Butyl-cyclohexyl)-phenyl]-2-carboxy-ethyl}-benzoicacid tert-butyl ester, was subjected to the procedures of steps 9-11 togive(R)-2-{4-[2-[4-(4-(trans)-tert-butyl-cyclohexyl)-phenyl]-2-(4′-chloro-2′-methyl-biphenyl-4-ylcarbamoyl)-ethyl]-benzoylamino}-ethanesulfonicacid.

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the claimed embodiments, and are not intended to limit thescope of what is disclosed herein. Modifications that are obvious topersons of skill in the art are intended to be within the scope of thefollowing claims. All publications, patents, and patent applicationscited in this specification are incorporated herein by reference intheir entireties as if each such publication, patent or patentapplication were specifically and individually indicated to beincorporated herein by reference.

What is claimed is:
 1. A compound of Formula I:

wherein R⁴⁴ is H, CH₃ or CH₃CH₂; R⁴⁵ is C₁₋₆-alkyl, alkenyl, alkoxy,C₃₋₆-cycloalkyl, C₄₋₈-cycloalkenyl, C₄₋₈-bicycloalkenyl, aryl orheteroaryl, any of which can be optionally substituted with one or moresubstituents selected from C₁₋₆alkyl, CF₃, F, CN or OCF₃; L is phenyl,indenyl, benzoxazol-2-yl, C₃₋₆-cycloalkyl, C₄₋₈-cycloalkenyl orC₄₋₈-bicycloalkenyl, any of which can be optionally substituted with oneor more substituents selected from F, Cl, CH₃, CF₃, OCF₃ or CN; and R⁴⁶represents one or more substituents selected from H, F, Cl, CH₃, CF₃,OCF₃ or CN; or a pharmaceutically acceptable salt, solvate, or prodrugthereof.
 2. The compound of claim 1, wherein: R⁴⁴ is H, CH₃ or CH₃CH₂;R⁴⁵ is C₁₋₆-alkyl, alkenyl, alkoxy, C₃₋₆-cycloalkyl, C₄₋₈-cycloalkenyl,C₄₋₈-bicycloalkenyl, aryl or heteroaryl, any of which can be optionallysubstituted with one or more substituents selected from C₁₋₆alkyl, CF₃,F, CN or OCF₃; L is phenyl, indenyl, benzoxazol-2-yl or4,4-dimethylcyclohexenyl, any of which can be optionally substitutedwith one or more substituents selected from F, Cl, CH₃, CF₃, OCF₃ or CN;and R⁴⁶ is H, F, Cl, CH₃, CF₃, OCF₃ or CN.
 3. The compound of claim 1,wherein L is phenyl, benzoxazol-2-yl or 4,4-dimethylcyclohexenyl, any ofwhich can be optionally substituted with one or more substituentsselected from F, Cl, CH3, CF3, OCF3 or CN.
 4. The compound of claim 1,wherein L is 4-chloro-2-methylphenyl, 4-methyl-2-benzoxazolyl,2,4,6-trimethylphenyl, 2-benzoxazolyl, 4-chloro-3-methylphenyl or4,4-dimethylcyclohexenyl.
 5. The compound of claim 1, wherein R⁴⁴ is Hor CH₃.
 6. The compound of claim 1, wherein R⁴⁴ is H.
 7. The compound ofclaim 1, wherein R⁴⁵ is attached to the 3 (meta) or 4 (para) position.8. The compound of claim 1, wherein R⁴⁵ is alkenyl, C₃₋₆-cycloalkyl,C₄₋₈-cycloalkenyl, C₄₋₈-bicycloalkenyl or phenyl, any of which can beoptionally substituted with one or more substituents selected fromC₁₋₆alkyl or CF₃.
 9. The compound of claim 1, wherein R⁴⁵ is substitutedwith one or more substituents independently selected from CH₃ and(CH₃)₃C—.
 10. The compound of claim 1, wherein R⁴⁵ is selected from(CH₃)₃CCH═CH—, t-butyl-cycloalkyl-, dimethyl-cycloalkyl-,t-butyl-cycloalkenyl-, dimethyl-cycloalkenyl-, bicycloalkenyl- ort-butyl-phenyl-.
 11. The compound of claim 1, wherein R⁴⁵ istrans-t-butylvinyl, cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl,4,4-dimethylcyclohexyl, cyclohex-1-enyl, (S)-4-t-butylcyclohex-1-enyl,(R)-4-t-butylcyclohex-1-enyl, 4,4-dimethylcyclohex-1-enyl,4,4-diethylcyclohex-1-enyl, 4,4-diethylcyclohexyl,4,4-dipropylcyclohex-1-enyl, 4,4-dipropylcyclohexyl, 4,4-dimethylcyclohexa-1,5-dienyl, (1R,4S)-1,7,7-trimethylbicyclo[2.2.1]3-heptyl-2-ene,(1R,4R)-1,7,7-trimethylbicyclo[2.2.1]2-heptyl-2-ene,2-methyl-4-chloro-phenyl, 2,4,6-trimethylphenyl or 4-t-butylphenyl. 12.The compound of claim 1, wherein is R⁴⁵ is trans-t-butylvinyl,cis-4-t-butylcyclohexyl, trans-4-t-butylcyclohexyl,4,4-dimethylcyclohexyl, (S)-4-t-butylcyclohex-1-enyl,(R)-4-t-butylcyclohex-1-enyl, 4,4-dimethylcyclohex-1-enyl,(1R,4R)-1,7,7-trimethylbicyclo[2.2.1]2-heptyl-2-ene or 4-t-butylphenyl.13. The compound of claim 1, wherein R⁴⁶ is H or CH₃.
 14. The compoundof claim 1, wherein the compound is selected from the group


15. The compound of claim 1, wherein the compound is:


16. A pharmaceutical composition comprising the compound of claim 1, andone or more pharmaceutically acceptable excipients or carriers.
 17. Thepharmaceutical composition of claim 16, further comprising a secondtherapeutic agent.
 18. The pharmaceutical composition of claim 16,wherein the second therapeutic agent is an antidiabetic agent.
 19. Apharmaceutical composition comprising the compound of claim 1 andcyclodextrin.
 20. A method of treating one or more symptoms of a diseaseresponsive to the modulation of a glucagon receptor in a subject,wherein the disease is diabetes, comprising administering to a subjectin need thereof the compound of claim
 1. 21. A method of treating one ormore symptoms of a disease responsive to a decrease in the hepaticglucose production or in the blood glucose level in a subject, whereinthe disease is diabetes, comprising administering to a subject in needthereof the compound of claim 1.